Oligosialic acid derivatives, methods of manufacture, and immunological uses

ABSTRACT

The invention relates to methods of producing, and compositions comprising, an isolated alpha (2→8) or (2→9) oligosialic acid derivative bearing a non-reducing end enriched for one or more de-N-acetyl residues and resistant to degradation by exoneuraminidase. A representative production method involves: (i) treating an alpha (2→8) or (2→9) oligosialic acid precursor having a reducing end and a non-reducing end with sodium borohydride under conditions for de-N-acetylating the non-reducing end; and (ii) isolating alpha (2→8) or (2→9) oligosialic acid derivative having one or more de-N-acetylated residues and a non-reducing end that is resistant to degradation by exoneuraminidase. Isolated alpha (2→8) or (2→9) oligosialic acid derivatives that comprise a non-reducing end de-N-acetyl residue are provided, as well as antibodies specific for the derivatives, compositions comprising the derivatives, kits, and methods of use including protection against and detection of  E. coli  K1 and  N. meningitidis  bacterial infection, and in diagnosing and treating cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit of U.S. provisional applicationSer. No. 60/958,342, filed Jul. 3, 2007, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grants no. AI64314awarded by the National Institute of Allergy and Infectious Diseases,and the National Institute of Health. The government has certain rightsin this invention.

TECHNICAL FIELD

This present disclosure relates to oligosialic acid derivatives,compositions containing the same, methods of their manufacture and use.

BACKGROUND

Sialic acid is an N— or O-substituted derivative of neuraminic acid. TheN-substituted versions generally bear either an acetyl or a glycolylgroup. In contrast, the O-substituted hydroxyl group may varyconsiderably, e.g., acetyl, lactyl, methyl, sulfate and phosphategroups. Polysialic acids are also quite common in which N-acetylneuraminic acid residues are linked via the C2 ketal OH to anothermolecule by a glycosidic bond, e.g., poly alpha (2→8) N-acetylneuraminic acid.

The sialic acids are biologically important carbohydrates found inorganisms ranging from bacteria to humans. They are common featuresdecorating the terminal ends of glycoproteins, glycans andglycosphingolipids, as well as other molecules. They mediate myriadnormal cellular activities. This includes stabilizing glycoconjugates incell membranes, regulating cell-cell interactions, acting as chemicalmessengers, regulating transmembrane receptor function, affectingmembrane transport, controlling the half-lives of circulatingglycoproteins and cells, and contributing to the permselectivity of theglomerular endothelium. See for review: Angata and Varki Chem. Rev.(2002) 102:439.

Given their prominent role in normal cellular activity, sialic acid andits derivatives have been used as markers for abnormal cellularprocesses such as cancer. (O'Kennedy et al., Cancer Lett., 1991 58:91;Vedralova et al. Cancer Lett. 1994 78:171; and Horgan et al., Clin.Chim. Acta., 1982 118:327; and Narayanan, S. Ann. Clin. Lab. Sci. 199424:376). For instance, cancer cells that can metastasize often havelarger amounts of sialic acid-modified glycoproteins, which may helpthem enter the blood stream. Also, the sialic acid of tumor cells ismodified in ways that differ from normal cells (Hakamori Cancer Res.1996, 56:5309, Dall'Olio Clin. Mol. Pathol. 1996, 49:M126, Kim and VarkiGlycoconj. J. 1997, 14:569).

One sialic acid derivative thought to be uncommon in normal cells, butpresent on cancer cells is de-N-acetyl sialic acid (Hanai et al J. Biol.Chem. 1988, 263:6296, Manzi et al J. Biol. Chem. 1990, 265:1309, Sjoberget al J. Biol. Chem. 1995, 270:2921, Chamas et al 1999, Cancer Res.59:1337; and Popa et al Glycobiology. 2007 17:367).

SEAM 3 is a murine monoclonal antibody that binds to poly alpha (2→8)N-acetyl neuraminic acid (polysialic acid or PSA) that containsde-N-acetyl residues (i.e., neuraminic acid) (Moe et al, Infect. Immun.,2005, 73:2123). SEAM 3 mediates bacteriolysis of Neisseria meningitidisgroup B (NmB) bacteria in the presence of exogenous complement andprovides passive protection in an in vivo infant rat model ofmeningococcal bacteremia. SEAM 3 also binds to PSA antigens expressed ina variety of human tumors resulting in arrest of cell growth and areduction in viability by inducing apoptosis and cell death.

Sodium borohydride is a reducing agent used to reduce aldehydes,ketones, imines, acid chlorides, and anhydrides. However, under mildbasic conditions, it has also been shown to de-N-acetylateglycosaminoglycuronans (Hirano et al, Connect Tissue Res, 1975, 3:73)and cleave amide bonds in polypeptides (Shimamura et al, Arch BiochemBiophys, 1984, 232:699). A product of the sodium borohydride reductionis sodium borate. Borates and boranes are known to form cyclic esterswith a variety of 1,2 or 1,3-diols in a 1:1 or 1:2 ratio. At pH<9borates form complexes with the alpha caroboxylate of N-acetylneuraminic acid and at pH >9 with the glycerol moiety at thenon-reducing end (Djanashvili et al, Chem Eur J, 2005, 11:4010).

Relevant Literature

Amino sugars, derivatives and related literature of interest arereported in the following U.S. Pat. Nos.: 4,021,542; 4,062,950;4,175,123; 4,216,208; 4,254,256; 4,314,999; 4,656,159; 4,713,374;4,797,477; 4,803,303; 4,840,941; 4,914,195; 4,968,786; 4,983,725;5,231,177; 5,243,035; 5,264,424; 5,272,138; 5,332,756; 5,667,285;5,674,988; 5,759,823; 5,962,434; 6,075,134; 6,110,897; 6,274,568;6,407,072; 6,458,937; 6,548,476; 6,697,251; 6,680,054; 6,936,701; and7,070,801, and in the following references: Angata and Varki Chem. Rev.2002, 102:439; Hakamori Cancer Res. 1996, 56:5309; Dall'Olio Clin. Mol.Pathol. 1996, 49:M126; Kim and Varki Glycoconj. J. 1997, 14:569; Hanaiet al J. Biol. Chem. 1988, 263:6296; Manzi et al J. Biol. Chem. 1990,265:1309; Sjoberg et al J. Biol. Chem. 1995, 270:2921; Chamas et alCancer Res. 1999, 59:1337; Popa et al Glycobiology. 2007 17:367; Kayseret al J. Biol. Chem. 1992 267:16934; Keppler et al Glycobiology 2001,11:1R; Luchansky et al Meth. Enzymol. 2003, 362:249; Oetke et al Eur. J.Biochem. 2001, 268:4553; Collins et al Glycobiology 2000, 10:11; andBardor et al J. Biol. Chem. 2005, 280:4228.

The antibody SEAM 3 is reported in Moe et al, Infect. Immun., 2005,73:2123. Sodium borohydride reactions and related are reported invarious references, such as Hirano et al, Connect Tissue Res, 1975,3:73; Shimamura et al, Arch Biochem Biophys, 1984, 232:699; andDjanashvili et al, Chem Eur J, 2005, 11:4010. See also See also US2007/0010482; U.S. application Ser. No. 11/645,255, filed Dec. 22, 2006;WO 2006/002402; and PCT application serial no. PCT/US2006/04885, filedDec. 22, 2006.

SUMMARY

The present invention relates to a method of producing, and compositionscomprising an isolated alpha (2→8) and alpha (2→9) oligosialic acidderivative bearing a reducing end that is enriched for one or morede-N-acetyl residues and resistant to degradation by exoneuraminidase.This includes compositions that are enriched with alpha (2→8) or (2→9)oligosialic acid derivatives that bear a non-reducing end enriched forde-N-acetyl residues and resistant to degradation by exoneuraminidase,as well as aggregates of the derivatives. A representative method ofproduction involves: (i) treating an alpha (2→8) or (2→9) oligosialicacid precursor having a reducing end and a non-reducing end with sodiumborohydride under conditions for de-N-acetylating the non-reducing end;and (ii) isolating alpha (2→8) or (2→9) oligosialic acid derivativehaving one or more de-N-acetylated residues and a non-reducing end thatis resistant to degradation by exoneuraminidase. An isolated alpha (2→8)or (2→9) oligosialic acid derivative produced by this method also isprovided, as well as antibodies specific for the derivative, andcompositions comprising the derivatives. The compositions comprising theaggregates are produced by the additional step of (iii) exposing theisolated alpha (2→8) or (2→9) oligosialic acid derivative to aggregatingconditions, so as to form the aggregate, and, optionally, isolating theaggregate.

Also provided are methods of inhibiting growth of a cancerous cell in asubject. This method involves administering to the subject an effectiveamount of a pharmaceutically acceptable formulation comprising anantibody specific for an alpha (2→8) or (2→9) oligosialic acidderivative bearing a reducing end enriched for de-N-acetyl residues andresistant to degradation by exoneuraminidase, where the administeringfacilitates reduction in viability of cancerous cells exposed to theantibody.

Also featured is a method of eliciting antibodies to bacteria (e.g., N.meningitidis, E. coli K1) and/or to cancerous cells that bear ade-N-acetylated sialic acid (deNAc SA) epitope. This method involvesadministering to a subject an immunogenic composition comprising anisolated alpha (2→8) or (2→9) oligosialic acid derivative bearing anon-reducing end enriched for de-N-acetyl residues and resistant todegradation by exoneuraminidase, where the administering is effective toelicit production of an antibody that specifically binds to a deNAc SAepitope of the bacterial or cancerous cell. This includes immunogeniccompositions that are enriched with alpha (2→8) or (2→9) oligosialicacid derivatives that bear a non-reducing end enriched for de-N-acetylresidues and resistant to degradation by exoneuraminidase. Also, theoligosialic acid derivatives and compositions can be used as a vaccineagainst bacteria with a de-N-acetyl sialic acid epitopes present intheir polysaccharide capsules, such as Neisseria, especially N.meningitidis, particularly N. meningitidis Groups B and C, and E. coliK1.

Also provided are methods of detecting a cancerous cell in a subject.This method involves contacting a biological sample obtained from asubject suspected of having cancer with an antibody specific for analpha (2→8) or (2→9) oligosialic acid derivative bearing a non-reducingend enriched for de-N-acetyl residues and resistant to degradation byexoneuraminidase, where binding of the antibody is indicative of thepresence of cancerous cells in the subject.

Kits containing one or more compositions of the present disclosure, aswell as those with instructions for use in a method of the presentdisclosure also are provided.

Accordingly, in one aspect the present disclosure provides methods ofproducing an isolated alpha (2→8) or (2→9) oligosialic acid derivativecomprising generating an alpha (2→8) or (2→9) oligosialic acidderivative having one or more de-N-acetylated residues by treating analpha (2→8) or (2→9) oligosialic acid precursor having a reducing endand a non-reducing end with sodium borohydride under conditions forde-N-acetylating the non-reducing end; and isolating the alpha (2→8) or(2→9) oligosialic acid derivative having (i) a degree of polymerizationof about 2-20, and (ii) one or more de-N-acetylated residues and anon-reducing end that is resistant to degradation by exoneuraminidase,whereby the isolated alpha (2→8) or (2→9) oligosialic acid derivative isproduced.

In related embodiments, the non-reducing end of the oligosialic acidderivative is a de-N-acetylated residue, In specific embodiments, thede-N-acetylated residue is neuraminic acid. In related embodiments, theoligosialic acid derivative comprises one or more N-acyl groups otherthan N-acetyl, In one embodiment, the N-acyl group is trichloroacetyl.In related embodiments, the oligosialic acid precursor is obtainablefrom acid hydrolysis of a polysialic acid polymer obtainable from abacterium selected from the group consisting of E. coli K1, Neisseriameningitidis serogroup B, and Neisseria meningitidis serogroup C.

In related embodiments, the oligosialic acid derivative has a degree ofpolymerization of about 2 to 10. In related embodiments, the oligosialicacid derivative is comprised as an isolated mixture of alpha (2→8) or(2→9) oligosialic acid chains, where in some embodiments the mixture ofalpha (2→8) or (2→9) oligosialic acid chains comprises shorter lengthchains and a ratio of sialic acid to de-N-acetylated sialic acid of 3:1and/or comprises longer length chains and a ratio of sialic acid tode-N-acetylated sialic acid of 10:1. In related embodiments, theisolated oligosialic acid derivative is capable of inhibiting SEAM 2,SEAM 3, or DA2 binding to dodecylamine N-propionyl NmB polysialic acidor N-propionyl NmB polysialic acid at an IC50 of less than about 0.1μg/ml.

In related embodiments, the method further comprises conjugating asecond molecule to the isolated alpha (2→8) or (2→9) oligosialic acidderivative, wherein the second molecule is selected from the groupconsisting of protecting group, amino acid, peptide, polypeptide, lipid,carbohydrate, nucleic acid and detectable label. In some embodiments,the second molecule is an immunomodulatory (e.g., a toxin or derivativethereof (e.g., a tetanus toxoid)).

In other embodiments, the method further comprises enriching for alpha(2→8) oligosialic acid derivative having a non-reducing end that isresistant to degradation by exoneuraminidase by exposure of the alpha(2→8) or (2→9) oligosialic acid derivative to exoneuraminidase. Inrelated embodiments, the oligosialic acid derivative is provided in anaggregate (e.g., an aggregate comprising microscopic particles).

The present disclosure also provides isolated alpha (2→8) or (2→9)oligosialic acid derivatives produced according to the methods of thepresent disclosure, as well as compositions comprising such compounds.

In another aspect, the present disclosure provides compositionscomprising an isolated alpha (2→8) or (2→9) oligosialic acid derivativeproduced according to the method of claim 1, wherein the isolated alpha(2→8) or (2→9) oligosialic acid derivative comprises as mixture ofoligosialic acid derivatives of variable chain lengths each having anon-reducing end de-N-acetyl residue.

In another aspect, the present disclosure provides compositionscomprising an alpha (2→8) or (2→9) oligosialic acid derivative having adegree of polymerization of about 2-20, and a reducing end and anon-reducing end, wherein the non-reducing end comprises ade-N-acetylated residue that is resistant to degradation byexoneuraminidase. In related embodiments, the non-reducing end of theoligosialic acid derivative is a de-N-acetylated residue. In relatedembodiments, the de-N-acetylated residue is neuraminic acid. In relatedembodiments, the oligosialic acid derivative comprises one or moreN-acyl groups other than N-acetyl. In related embodiments, the reducingend of the isolated oligosialic acid derivative is reduced. In relatedembodiments, the oligosialic acid is obtainable from a polysialic acidpolymer obtainable from a bacterium selected from the group consistingof E. coli K1, Neisseria meningitidis serogroup B, and Neisseriameningitidis serogroup C. In further related embodiments, 25 theoligosialic acid derivative comprises a degree of polymerization ofabout 2 to 10.

In related embodiments, the oligosialic acid derivative is comprised asan isolated mixture of oligosialic acid chains where, for example, themixture of oligosialic acid chains comprises shorter length chains and aratio of sialic acid to de-N-acetylated sialic acid of about 3:1 and/orthe mixture of oligosialic acid chains comprises longer length chainsand a ratio of sialic acid to de-N-acetylated sialic acid of about 10:1.

In related embodiments, the oligosialic acid derivative comprises aconjugate, e.g., where the oligosialic acid derivative is conjugated toone or more second molecules selected from the group consisting ofprotecting group, amino acid, peptide, polypeptide, lipid, carbohydrate,nucleic acid and detectable label. In some embodiments, the secondmolecule is an immunomodulatory (e.g., a a toxin or derivative thereof(e.g., a tetanus toxoid)). In related embodiments, the oligosialic acidderivative is comprised as a formulation containing one or moreimmunogenic excipients. In related embodiments, the oligosialic acidderivative is capable of inhibiting SEAM 2, SEAM 3 and DA2 binding tododecylamine N-propionyl NmB polysialic acid or N-propionyl NmBpolysialic acid at an IC50 of less than about 0.1 μg/ml. In relatedembodiments, the isolated alpha (2→8) or (2→9) oligosialic acidderivative comprises as mixture of oligosialic acid derivatives ofvariable chain lengths that have a non-reducing end enriched withde-N-acetyl residues. In related embodiments, the oligosialic acidderivative is provided in an aggregate (e.g., aggregates comprisingmicroscopic particles).

In another aspect, the present disclosure provides isolated antibodiesspecific for an alpha (2→8) or (2→9) oligosialic acid derivative thatcomprises a non-reducing end enriched for one or more de-N-acetylatedresidues and is resistant to degradation by exoneuraminidase. In relatedembodiments, the antibody is specific for alpha (2→8) or (2→9)oligosialic acid derivative in an aggregate, e.g., aggregates comprisinga microscopic particle. In related embodiments, the antibody is capableof complement mediated bacteriolysis and opsonophagocytosis of Neisseriameningitidis group B (NmB) and group C (NmC) bacteria. In relatedembodiments, the antibody is capable of binding neuraminicacid-containing antigens expressed by dividing or non-dividing JurkatT-cell leukemia cells, and in further related embodiments, the antibodybinds the non-dividing Jurkat T-cell leukemia cells better than SEAM 3.In related embodiments, the antibody is of mouse origin. In furtherrelated embodiments, the antibody is specific for non-reducing endde-N-acetyl sialic acid residue.

In specific embodiments, the antibody is a monoclonal antibody having alight and heavy chain variable complementarity determining regionpolypeptide sequence as depicted in FIGS. 19 and 20, and in specificembodiments is a monoclonal antibody having a complementaritydetermining region (CDR) polypeptide sequence selected from a CDRpolypeptide sequence depicted in FIG. 19 or 20. In related embodiments,the monoclonal antibody is a humanized monoclonal antibody.

In related aspects, the present disclosure provides methods of detectinga cancerous cell in a subject, the method comprising contacting abiological sample obtained from a subject suspected of having cancerwith an antibody according to the present disclosure, wherein thebinding of the antibody is indicative of the presence of cancerous cellsin the subject. In further related aspects, the present disclosureprovides methods of inhibiting growth of a cancerous cell in a subjectcomprising administering to the subject an effective amount of apharmaceutically acceptable formulation comprising an antibody of thepresent disclosure, wherein the administering facilitates reduction inviability of cancerous cells exposed to the antibody. In still otherrelated aspects, the present disclosure provides methods of elicitingantibodies in a subject, where the antibodies specifically bind abacteria comprising a de-N-acetylated sialic acid (deNAc SA) epitopecomprising administering to a subject an immunogenic compositioncomprising an alpha (2→8) or (2→9) oligosialic acid derivative having adegree of polymerization of about 2 to 20, and a reducing end and anon-reducing end, wherein the non-reducing end is enriched for one ormore de-N-acetylated residues and resistant to degradation byexoneuraminidase, and wherein the administering is effective to elicitproduction of an antibody that specifically binds a deNAc SA epitope ofa bacteria. In related embodiments, the bacteria is Neisseriameningitidis group B, Neisseria meningitidis group C, or Escherichiacoli K1.

In another aspect, the present disclosure provides methods of elicitingantibodies to a cancerous cell comprising a de-N-acetylated sialic acid(deNAc SA) epitope in a subject comprising administering to a subject animmunogenic composition comprising an alpha (2→8) or (2→9) oligosialicacid derivative having a degree of polymerization of about 2 to 20, anda reducing end and a non-reducing end, wherein the non-reducing end isenriched for one or more de-N-acetylated residues and resistant todegradation by exoneuraminidase, and wherein the administering iseffective to elicit production of an antibody that specifically binds adeNAc SA epitope of the cancerous cell. In related embodiments, thecancer is a melanoma, a lymphoma, or a neuroblastoma.

In related embodiments, the alpha (2→8) or (2→9) oligosialic acidderivative of the immunogenic composition is prepared by selectivede-acetylation of non-reducing end residue by sodium borohydridereduction. In related embodiments, the alpha (2→8) or (2→9) oligosialicacid derivative is a conjugate.

In related embodiments, the alpha (2→8) or (2→9) oligosialic acidderivative is administered by infusion or by local injection. In relatedembodiments, administering can be prior to surgical intervention toremove cancerous cells, at the time of or after surgical intervention toremove cancerous cells, and/or administered in conjunction with at leastone of an immunotherapy, a cancer chemotherapy or a radiation therapy.In related embodiments, the isolated alpha (2→8) or (2→9) oligosialicacid derivative comprises an aggregate of the polysialic acid derivative(e.g., aggregates comprising microscopic particles). In relatedembodiments,

In another aspect, the present disclosure provides methods of producingan aggregate comprising an alpha (2→8) or (2→9) oligosialic acidderivative comprising admixing one or more alpha (2→8) or (2→9)oligosialic acid derivatives under aggregating conditions so as to forman aggregate. In related embodiments, the aggregating conditions isheating (e.g., heating from 30° C. to 70° C.) or the addition of anaggregating excipient (e.g., aluminum hydroxide). In relatedembodiments, the aggregate is a particle, e.g., a microscopic particle.In related embodiments, the polysialic acid derivative has a mixture ofN-acetyl and de-N-acetyl residues and is resistant to degradation byexoneuraminidase.

In other aspects, the present disclosure provides, vaccine compositionscomprising an isolated alpha (2→8) or (2→9) oligosialic acid derivativehaving (i) a degree of polymerization of about 2-20, (ii) an IC50 ofless than about 0.1 μg/ml for inhibiting SEAM 2, SEAM 3 or DA2 antibodybinding to dodecylamine N-propionyl NmB polysialic acid or N-propionylNmB polysialic acid, and (iii) a non-reducing end de-N-acetyl residuethat is resistant to degradation by exoneuraminidase. In relatedembodiments, the derivative comprises one or more N-trichloroacetylsialic acid residues. In related embodiments, the derivative comprisesone or more N-propionyl sialic acid residues. In related embodiments,the derivative has a degree of polymerization of about 2-10 and/or about2-6. In related embodiments, the derivative is selected from the groupconsisting of dimer, trimer and tetramer. In related embodiments, thederivative comprises a conjugate (e.g., is conjugated to a secondmolecule comprising an immunomodulatory, such as a toxin or derivativethereof (e.g., a tetanus toxoid)). Exemplary immunomodulatory conjugatesof derivatives of the present disclosure include NPrSia-TT, OS-TT, andTcAc-TT.

In another aspect, the present disclosure provides vaccine compositionscomprising an isolated alpha (2→8) or (2→9) oligosialic acid derivativehaving (i) a degree of polymerization of about 2-20, (ii) a de-N-acetylsialic acid content of about 50% to 98%, and (iii) a non-reducing endde-N-acetyl residue that is resistant to degradation byexoneuraminidase. In related embodiments, the derivative has ade-N-acetyl sialic acid content of about 88% to 98%. In relatedembodiments, the derivative has a degree of polymerization of about 2-10and/or about 2-6. In related embodiments, the derivative is a dimer,trimer or tetramer. In related embodiments, the derivative vaccinecomprises a conjugate (e.g., is conjugated to a second moleculecomprising an immunomodulatory, such as a toxin or derivative thereof(e.g., a tetanus toxoid)). Exemplary immunomodulatory conjugates ofderivatives of the present disclosure include NPrSia-TT, OS-TT, andTcAc-TT. In related embodiments, the derivative is DeNAc-TT.

In aspects relating to vaccine compositions, related embodiments includevaccine compositions comprising an adjuvant. Furthermore, relatedembodiments of such aspects include vaccine compositions wherein thederivative is present in the composition in an effective amount toelicit production of an antibody that specifically binds a deNAc SAepitope of a cell in a subject administered the vaccine composition.

Other features of the invention are described herein, and will also bereadily apparent to the ordinarily skilled artisan upon reading thepresent disclosure.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts the results of anion exchange chromatography of sodiumborohydride-treated oligosialic acid (OS).

FIG. 2 depicts the results of analysis of column fractions from anionexchange chromatography by high performance anion exchangechromatography with pulsed ampermetric (HPAC-PAD) detection.

FIG. 3 is a Western blot of the OS-tetanus toxoid (OS-TT) conjugatevaccine using SEAM 3 as the primary detecting antibody.

FIG. 4 summarizes the OS-specific antibody titers resulting fromimmunizing CD1 mice with the OS-TT conjugate vaccine.

FIG. 5 shows the measurement of complement factor deposition on live NmBbacteria by flow cytometry mediated by pooled antisera from miceimmunized with the OS-TT conjugate vaccine.

FIG. 6 is a fluorescence micrograph of bacteria show deposition ofcomplement factors on the cells mediated by pooled antisera from miceimmunized with the OS-TT conjugate vaccine.

FIG. 7 is a bar graph showing the effect of pooled antisera from miceimmunized with the OS-TT conjugate vaccine on the growth of N.meningitidis serogroup B (NmB) bacteria in an ex vivo human blood modelof meningococcal bacteremia.

FIG. 8 shows the measurement of antibodies elicited by immunization ofCD1 mice with the OS-TT conjugate vaccine binding to Jurkat T-cellleukemia cells by flow cytometry.

FIG. 9 is a Western blot of the PS-tetanus toxoid conjugate vaccinesusing SEAM 18, SEAM 2, or SEAM 3 as primary detecting antibodies.

FIG. 10 summarizes the ELISA titers of antiserum pools from CD1 miceimmunized with 1, 2, or 3 doses of 2 μg or 10 μg of total sialic acid ofeach of the PS-tetanus toxoid conjugate vaccines. The upper panel showsthe titers against the homologous PS antigens and lower panel shows thetiters against the DeNAc antigen.

FIG. 11 summarizes the results of measuring control and antiserumantibody binding to N. meningtidis group B strain NMB by flow cytometry.Panel A shows of binding of IgG and IgM antibodies for all antiserumpools. Panel B shows binding for each IgG subclass of the 10 μg dose3^(rd) injection DeNAc antiserum pool.

FIG. 12 shows the ability of vaccine elicted antibodies to activatecomplement protein deposition on N. meningitidis group B strain NMB(panel A) and group C strain 4243.

FIG. 13 shows the results of evaluating the ability of PS-conjugatevaccine elicited antisera to passively protect in an infant rat model ofmeningococcal bacteremia. The upper panel shows the results forchallenge with N. meningitidis group B strain M986 and the lower panelfor group C strain 4243.

FIG. 14 is a bar graph showing the effect of pooled antisera from miceimmunized with the PS-conjugate vaccines on the growth of N.meningitidis group B strain NZ98/254 bacteria in an ex vivo human bloodmodel of meningococcal bacteremia.

FIG. 15 is a bar graph showing the measurement of antibodies elicted byimmunization of CD1 mice with the PS-conjugate vaccines binding toJurkat T-cell leukemia cells by flow cytometry.

FIG. 16 is a bar graph showing the measurement of the ability ofantibodies elicted by immunization of CD1 mice with the PS-conjugatevaccines to activate deposition of human complement proteins on JurkatT-cell leukemia, SK-MEL 28 melanoma, and CHP-134 neuroblastoma cells byflow cytometry.

FIG. 17 is a bar graph showing the measurement of the ability ofantibodies elicted by immunization of CD1 mice with the PS-conjugatevaccines to decrease the viability of Jurkat T-cell leukemia cells.

FIG. 18 is a set of light micrographs of immunohistochemical staining ofnormal ovary and a primary ovarian tumor with polyclonal antiseraelicited by immunization of CD1 mice with tetanus toxoid carrier proteinor TcAc-tetanus toxoid vaccine.

FIG. 19 shows the relationship of the DNA sequence and correspondingamino acid sequence translation of the DA2 heavy chain variable regiongene to variable region framework and CDRs as defined by InternationalImmunogenetics Information System (IMGT) definitions (Lefranc et al.IMGT, the international ImMunoGeneTics information system®. Nucl. AcidsRes., 2005, 33, D593-D597).

FIG. 20 shows the relationship of the DNA sequence and correspondingamino acid sequence translation of the DA2 light chain variable regiongene to variable region framework and CDRs as defined by InternationalImmunogenetics Information System (IMGT) definitions (Lefranc et al.IMGT, the international ImMunoGeneTics information system®. Nucl. AcidsRes., 2005, 33, D593-D597).

FIG. 21 is a bar graph showing the measurement of the ability of themonoclonal antibody to decrease the viability of Jurkat T-cell leukemiacells compared to an irrelevant IgM control antibody.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

It was discovered that oligosialic acid derivatives bearing anon-reducing end de-N-acetyl residue elicit antibodies highly specificfor E. coli K1, N. meningitidis, and cancer cells expressing thisimmunodominant epitope. The derivatives of the present disclosure can beproduced by treatment of oligomers of sialic acid (N-acetyl neuraminicacid oligosaccharides (OS) ranging from dimers to multimers, e.g., of upto about 100 monomer units in length) with sodium borohydride underconditions that produce oligosialic acid product having derivatives witha non-reducing end that is enriched with one or more de-N-acetylresidues and resistant to treatment with exoneuraminidase. The productsof the sodium borohydride reaction are highly reactive with antibodiessuch as SEAM 3. It also has been discovered that the OS derivativescontaining the minimal features necessary for activity have (i) a degreeof polymerization of about 2-20, particularly sub-ranges thereof ofdimer, trimer and/or tetramer, and (ii) an immunodominant non-reducingend de-N-acetyl residue that is resistant to degradation byexoneuraminidase. Compounds have been produced with these and otherfeatures that can be exploited for a given end use. When conjugated to acarrier protein and used to immunize mice, the non-reducing end enrichedde-N-acetylated oligosaccharide products elicit antibodies that areprotective against N. meningitidis serogroup B (NmB), as well as otherbacteria expressing the immunodominant non-reducing end de-N-acetylresidue. They also bind to neuraminic acid-containing polysialic acid(PSA) antigens expressed by tumor cells. The disclosure is further basedon the discovery that the OS derivatives can be composed of alpha (2→8)and/or alpha (2→9) linked oligosialic acid material. The disclosure alsois based on the discovery that aggregates of the OS derivatives are morereadily taken up by cells and expressed on the cell surface as comparedto the corresponding non-aggregated OS derivative. The aggregates can beexploited in conjunction with or in the absence of carrier protein toelicit a strong T-cell dependent immune response. Antibody specific forthe immunodominant non-reducing end de-N-acetyl residue epitope havealso been discovered. The data support broad use of the methods andcompositions, including the diagnosis and treatment of multiple types ofcancer in humans, as well as diagnosis of and protection against diseasecaused by bacteria such as E. coli K1 and Neisseria.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, exemplarymethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantigen” includes a plurality of such antigens and reference to “thepeptide” includes reference to one or more peptides and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

When describing the compositions, pharmaceutical formulations containingsuch, and methods of producing and using such compositions, thefollowing terms have the following meanings unless otherwise indicated.It should also be understood that any of the moieties defined forthbelow may be substituted with a variety of substituents, and that therespective definitions are intended to include such substituted moietieswithin their scope.

The term “amino sugar” refers to a sugar or saccharide that contains anamino group in place of a hydroxyl group. Derivatives of aminocontaining sugars, such as N-acetyl-glucosamine, N-acetyl mannosamine,N-acetyl galactosamine, N-acetyl neuraminic acid and sialic acids ingeneral are examples of amino sugars.

The term “analog” or “analogue” refers to without limitation anycompound which has structural similarity to the compounds of the presentdisclosure and would be expected, by one skilled in the art, to exhibitthe same or similar utility as the claimed and/or referenced compounds.

The term “carrier” as used in the context of a carrier conjugated to analpha (2→8) oligosialic acid derivative generally refers to a peptide orprotein carrier, such as an antibody or antibody fragment. “Carrier”encompasses peptides or proteins that enhance immunogenicity of acompound.

The term “cell surface antigen” (or “cell surface epitope”) refers to anantigen (or epitope) on surface of a cell that is extracellularlyaccessible at any cell cycle stage of the cell, including antigens thatare predominantly or only extracellularly accessible during celldivision. “Extracellularly accessible” in this context refers to anantigen that can be bound by an antibody provided outside the cellwithout need for permeabilization of the cell membrane.

The term “chemotherapy” as used herein refers to use of an agent (e.g.,drug, antibody, etc.), particularly an agent(s) that is selectivelydestructive to a cancerous cell, in treatment of a disease, withtreatment of cancer being of particular interest.

A “cancer cell” as used herein refers to a cell exhibiting a neoplasticcellular phenotype, which may be characterized by one or more of, forexample, abnormal cell growth, abnormal cellular proliferation, loss ofdensity dependent growth inhibition, anchorage-independent growthpotential, ability to promote tumor growth and/or development in animmunocompromised non-human animal model, and/or any appropriateindicator of cellular transformation. “Cancer cell” may be usedinterchangeably herein with “tumor cell”, and encompasses cancer cellsof a solid tumor, a semi-solid tumor, a primary tumor, a metastatictumor, and the like.

The term “conjugated” generally refers to a chemical linkage, eithercovalent or non-covalent, usually covalent, that proximally associatesone molecule of interest with second molecule of interest.

The term “de-N-acetyl sialic acid antigen” (which may also be referredto as “de-N-acetylated sialic acid antigen” or “deNAc SA antigen”)refers to a compound having or mimicking a deNAc sialic acid epitope(deNAc SA epitope), which epitope is minimally defined by a dimer ofresidues of sialic acid or sialic acid derivative, where the dimercontains at least one de-N-acetylated sialic acid residue adjacent anN-acylated (e.g., acetylated or propionylated) sialic acid residue or asialic acid derivative residue. Examples of de-N-acetyl sialic acidantigens are provided in the present disclosure, and include, withoutlimitation, de-N-acetylated polysaccharide derivatives (“PSderivatives”), de-N-acetylated gangliosides, and de-N-acetylatedderivatives of a sialic-acid modified protein, particularly asialic-acid modified protein that is accessible at an extracellularsurface of a mammalian cell, particularly a human cell, moreparticularly a cancer cell, particularly a human cancer cell. DeNAc SAepitopes are also present in polysaccharide capsules of Neisseria,especially N. meningitidis, particularly N. meningitidis Group B, and E.coli K1. It should be noted that description of a deNAc SA antigen as aderivative of a starting molecule (e.g., PS derivative or gangliosidederivative) is not meant to be limiting as to the method of productionof the de-N-acetyl sialic acid antigen, but rather is meant as aconvenient way to describe the structure of the exemplary deNAc SAantigen.

The term “derivative” refers to without limitation any compound whichhas a structure derived from the structure of the compounds of thepresent disclosure and whose structure is sufficiently similar to thosedisclosed herein and based upon that similarity, would be expected, byone skilled in the art, to exhibit the same or similar activities andutilities as the claimed and/or referenced compounds.

The term “effective amount” of a compound as provided herein is intendedto mean a non-lethal but sufficient amount of the compound to providethe desired utility. For instance, for eliciting an immune response in asubject to generate anti-deNAc SA antibodies, the effective amount isthe amount which elicits a useful antibody response, e.g., so as toprovide for production of antibodies that can be subsequently isolated(e.g., as in monoclonal antibody production) or to provide for aclinically meaningful immune response in a subject against a bacteria(e.g., as in the context of prophylactic or therapeutic immunizationagainst a disease caused by Neisseria or E. coli K1) or by a cancercharacterized by a deNAc SA epitope. As will be pointed out below, theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe condition or disease that is being treated, the particular compoundused, its mode of administration, and the like. Thus, it is not possibleto specify an exact “effective amount.” However, an appropriateeffective amount may be determined by one of ordinary skill in the artusing only routine experimentation.

The term “immunotherapy” refers to treatment of disease (e.g., Neisseriaor E. coli K1 bacterial infection, cancer) by modulating an immuneresponse to a disease antigen. In the context of the presentapplication, immunotherapy refers to providing an antibacterial and/oranti-cancer immune response in a subject by administration of anantibody (e.g., a monoclonal antibody) and/or by administration of anantigen that elicits an anti-tumor antigen immune response in thesubject.

The term “inactivation” of a cell is used herein to indicate that thecell has been rendered incapable of cell division to form progeny. Thecell may nonetheless be capable of response to stimulus and/orbiosynthesis for a period of time, e.g., to provide for production of acell surface molecule (e.g., cell surface protein or polysaccharide).

The term “in combination with” as used herein refers to uses where, forexample, a first therapy is administered during the entire course ofadministration of a second therapy; where the first therapy isadministered for a period of time that is overlapping with theadministration of the second therapy, e.g. where administration of thefirst therapy begins before the administration of the second therapy andthe administration of the first therapy ends before the administrationof the second therapy ends; where the administration of the secondtherapy begins before the administration of the first therapy and theadministration of the second therapy ends before the administration ofthe first therapy ends; where the administration of the first therapybegins before administration of the second therapy begins and theadministration of the second therapy ends before the administration ofthe first therapy ends; where the administration of the second therapybegins before administration of the first therapy begins and theadministration of the first therapy ends before the administration ofthe second therapy ends. As such, “in combination” can also refer toregimen involving administration of two or more therapies. “Incombination with” as used herein also refers to administration of two ormore therapies which may be administered in the same or differentformulations, by the same or different routes, and in the same ordifferent dosage form type.

The term “isolated” is intended to mean that a compound is separatedfrom all or some of the components that accompany it in nature.“Isolated” also refers to the state of a compound separated from all orsome of the components that accompany it during manufacture (e.g.,chemical synthesis, recombinant expression, culture medium, and thelike).

The term “monoclonal antibody” refers to an antibody composition havinga homogeneous antibody population. The term is not limited by the mannerin which it is made. The term encompasses whole immunoglobulinmolecules, as well as Fab molecules, F(ab′)2 fragments, Fv fragments,single chain fragment variable displayed on phage (scFv), fusionproteins comprising an antigen-binding portion of an antibody and anon-antibody protein, and other molecules that exhibit immunologicalbinding properties of the parent monoclonal antibody molecule. Methodsof making polyclonal and monoclonal antibodies are known in the art anddescribed more fully below.

The term “non-reducing end” of an oligo or polysaccharide chain isintended the end portion of the chain bearing the non-reducing glycosylresidue.

The term “reducing end” of an oligo or polysaccharide chain is intendedthe end portion of the chain bearing the reducing glycose residue. Thisis the end of the chain that, when bearing a free anomeric carbon inbasic solution, is capable of forming an aldehyde or ketone.

The term “enriched” as used herein refers to a compound or compositionthat has an increase in the proportion of a desirable property orelement. For example, an alpha (2→8) oligosialic acid derivative that is“enriched” for de-N-acetylation at a non-reducing end is an alpha (2→8)oligosialic acid derivative in which the de-N-acetylated residues areprimarily present, including only present, at a non-reducing end,including the non-reducing terminal end. A composition is “enriched” foralpha (2→8) oligosialic acid derivatives having de-N-acetylatednon-reducing ends where the majority of alpha (2→8) oligosialic acidderivatives in the composition (e.g., more than 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more up to 100%) have a de-N-acetylatedresidue at a non-reducing end, particularly at a non-reducing terminalend.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable, i.e., the material is of amedically acceptable quality and composition that may be administered toan individual along with the selected active pharmaceutical ingredientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The term “pharmaceutically acceptable excipient” as used herein refersto any suitable substance which provides a pharmaceutically acceptablevehicle for administration of a compound(s) of interest to a subject.“Pharmaceutically acceptable excipient” can encompass substancesreferred to as pharmaceutically acceptable diluents, pharmaceuticallyacceptable additives and pharmaceutically acceptable carriers.

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;fusion proteins with detectable fusion partners, e.g., fusion proteinsincluding as a fusion partner a fluorescent protein, β-galactosidase,luciferase, etc.; and the like. Polypeptides may be of any size, and theterm “peptide” refers to polypeptides that are 8-50 residues (e.g., 8-20residues) in length.

The term “purified” is intended to mean a compound of interest has beenseparated from components that accompany it in nature and provided in anenriched form. “Purified” also refers to a compound of interestseparated from components that can accompany it during manufacture(e.g., in chemical synthesis, recombinant expression, culture medium,and the like) and provided in an enriched form. Typically, a compound issubstantially pure when it is at least 50% to 60%, by weight, free fromorganic molecules with which it is naturally associated or with which itis associated during manufacture. Generally, the preparation is at least75%, more usually at least 90%, and generally at least 99%, by weight,of the compound of interest. A substantially pure compound can beobtained, for example, by extraction from a natural source (e.g.,bacteria), by chemically synthesizing a compound, or by a combination ofpurification and chemical modification. A substantially pure compoundcan also be obtained by, for example, enriching a sample having acompound that binds an antibody of interest. Purity can be measured byany appropriate method, e.g., chromatography, mass spectroscopy, HPLCanalysis, etc.

The term “SEAM 3-reactive antigen” refers to an antigen having anepitope that is specifically bound by the monoclonal antibody (mAb) SEAM3 (ATCC Deposit No. HB-12170). The term “DA2-reactive antigen” refers toan antigen having an epitope that is specifically bound by themonoclonal antibody (mAb) DA2 (described herein). The monoclonalantibody DA2 is highly specific for any non-reducing end neuraminic acidresidue, regardless of the adjacent residue or glycosidic linkage.Exemplary SEAM 3 and/or DA2-reactive antigens are provided in theworking examples. The antibodies disclosed herein generated by anoligosialic acid-conjugate vaccine (OS-conjugate vaccine) also includethose that have antigen specificity other than binding to an epitopebound by SEAM 3, and may bind the same or different antigen, but doesnot bind normal PSA control (i.e., normal polysialic acid that is devoidof de-N-acetyl residues), and binds to OS-conjugate vaccine-generatedantigen better than SEAM 3 relative to normal PSA control. For example,DA2 binds the immunodominant de-N-acetyl residue epitope better thanSEAM 3, and as noted above, recognizes with high specificity anynon-reducing end neuraminic acid residue, regardless of the adjacentresidue or glycosidic linkage.

By “degree of polymerization” or Dp is intended the number of repeatunits in an average polymer chain. Chain length can be reported inmonomer units, as molecular weight, or both.

The term “subject” is intended to cover humans, mammals and otheranimals which contain polysialic acid in any fashion. The terms“subject,” “host,” “patient,” and “individual” are used interchangeablyherein to refer to any mammalian subject for whom diagnosis or therapyis desired, particularly humans. Other subjects may include cattle,dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

In the context of cancer therapies and diagnostics described herein,“subject” or “patient” is used interchangeably herein to refer to asubject having, suspected of having, or at risk of developing a tumor,where the cancer is one associated with cancerous cells expressing ade-N-acetyl sialic acid antigen. Samples obtained from such subject arelikewise suitable for use in the methods of the present disclosure.

As used herein, the terms “determining,” “measuring,” and “assessing,”and “assaying” are used interchangeably and include both quantitativeand qualitative determinations.

It is further noted that the claims may be drafted to exclude anyoptional or alternative element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed. To the extent a definitionof a term set out in a document incorporated herein by referenceconflicts with the definition of a term explicitly defined herein, thedefinition set out herein controls.

Exemplary methods and compositions employable therein are describedfirst in greater detail, followed by a review of the various specificcompositions, formulations, kits and the like that may find use in themethods of the present disclosure, as well as a discussion ofrepresentative applications in which the methods and compositions of thepresent disclosure find use.

Methods of Production and Compositions

As summarized above, the present disclosure provides oligosialic acid(OS) derivatives bearing a non-reducing end de-N-acetyl residue thatelicit antibodies highly specific for E. coli K1, N. meningitidis, andcancer cells expressing this immunodominant epitope. The OS derivativesgenerally have (i) a degree of polymerization of about 2-20,particularly sub-ranges thereof of dimer, trimer and/or tetramer, and(ii) an immunodominant non-reducing end de-N-acetyl residue that isresistant to degradation by exoneuraminidase. The OS derivatives can becomposed of alpha (2→8) and/or alpha (2→9) linked oligosialic acidmaterial. Also provided are methods of producing an isolated alpha (2→8)or alpha (2→9) oligosialic acid derivative bearing a non-reducing endenriched for one or more de-N-acetyl residues and that is resistant todegradation by exoneuraminidase. This also includes a method for theproduction of aggregates of the oligosialic acid derivatives, as well ascompositions and pharmaceutical formulations thereof. The products ofthe process find use in the production of oligosialic acid derivativecompositions and antibodies specific for the derivatives for a varietyof applications, including use in various methods of treating a hostsuffering from disease or condition in need thereof, e.g., for thediagnosis and/or treatment of a subject using an immunogenic and/orvaccine composition and/or antibody derived from an oligosialic acidderivative of the present disclosure (as described in greater detailbelow).

As noted above, compositions of the present disclosure include anisolated alpha (2→8) or (2→9) oligosialic acid derivative having adegree of polymerization of about 2-20, and a non-reducing endcomprising a de-N-acetylated residue that is resistant to degradation byexoneuraminidase. Thus, for example, an oligosialic acid derivativesinclude those that comprise a polymer of sialic and neuraminic acidmonomers joined essentially through alpha (2→8) or alpha (2→9)glycosidic linkages. One or more of the sialic and neuraminic acidmonomers of a polysialic acid may be modified or conjugated to a secondmolecule, such as a partially or fully O-acetylated monomer of sialicand/or neuraminic acid. In general, the oligosialic acid is obtainablefrom a polysialic acid polymer, for example, from a bacterium such as E.coli K1, Neisseria meningitidis serogroup B, and Neisseria meningitidisserogroup C, or can be synthesized de novo by other methods as describedin more detail below.

In some embodiments, the oligosialic acid derivative is comprisedessentially of a mixture of N-acetyl and de-N-acetyl sialic acidresidues, such as N-acetyl neuraminic acid and de-N-acetyl neuraminicacid. In other embodiments, the oligosialic acid derivative comprisesone or more N-acyl groups other than N-acetyl, such as a trichloroacetylor propionyl group.

In particular embodiments, the oligosialic acid derivative comprises aparticular degree of polymerization, such as degree of polymerization ofabout 2 to 10, and sub-ranges thereof of dimer, trimer and/or tetramer.In some embodiments, the oligosialic acid derivative is comprised as anisolated mixture of oligosialic acid chains. In particular embodiments,the mixture of oligosialic acid chains comprise shorter length chains.In other embodiments, the mixture of oligosialic acid chains compriseslonger length chains. In certain embodments, the oligosialic acidderivative is purified so as to be enriched for the desired mixture ofchains, or is purified to consist essentially of a single species ofchain.

The compositions of the present disclosure include an effective amountof the oligosialic acid derivative to achieve the desired end result.For example, the compositions generally include an effective amount ofthe derivative to elicit production of an antibody that specificallybinds a deNAc SA epitope of a cell in a subject administered the vaccinecomposition. In some instances, the antibody is immunoglobulin G (IgG),which may include a predominant response in which one or more subclassesof IgG are elicited, such as IgG1, IgG2, IgG3, and IgG4. Of specificinterest are IgG3 and IgG1. In other embodiments, the compositions ofthe present disclosure include an isolated oligosialic acid derivativein which the composition is substantially free of other oligosialic acidmaterial, and in certain embodiments, is substantially free ofoligosialic acid derivative having an N-acetyl sialic acid residue.

A featured aspect is a vaccine composition that includes an oligosialicacid derivative of the present disclosure. The vaccine compositions mayinclude oligosialic acid that is conjugated or unconjugated, and mayoptionally further include an adjuvant to enhance the effectiveness ofthe vaccine composition.

In certain embodiments, the vaccine composition comprises an isolatedalpha (2→8) or (2→9) oligosialic acid derivative having (i) a degree ofpolymerization of about 2-20, (ii) an IC50 of less than about 0.1 μg/mlfor inhibiting SEAM 2, SEAM 3, or DA2 antibody binding to dodecylamineN-propionyl NmB polysialic acid or N-propionyl NmB polysialic acid, and(iii) a non-reducing end de-N-acetyl residue that is resistant todegradation by exoneuraminidase. A featured aspect of this embodiment iswhere the oligosialic acid derivative comprises one or more N-acylgroups other than N-acetyl, such as one or more N-trichloroacetyl sialicacid residues or N-propionyl sialic acid residues.

In other embodiments, the vaccine composition comprise an isolated alpha(2→8) or (2→9) oligosialic acid derivative having (i) a degree ofpolymerization of about 2-20, (ii) a de-N-acetyl sialic acid content ofabout 50% to 98%, and (iii) a non-reducing end de-N-acetyl residue thatis resistant to degradation by exoneuraminidase. In this particularembodiment, the oligosialic acid derivative is generally composedessentially of a mixture of N-acetyl sialic acid and de-N-acetyl sialicacid residues. A featured aspect of this embodiment it where theoligosialic acid derivative has a de-N-acetyl sialic acid content ofabout 88% to 98%, usually about 95% to about 98%.

The vaccine compositions of specific interest include those where theoligosialic acid derivative has a degree of polymerization of about2-10, about 2-6, or less, and sub-ranges thereof of dimer, trimer andtetramer. In some embodiments, the vaccine compositions are composed ofessentially a single species of oligosialic acid derivative, forexample, dimer, trimer or tetramer.

The vaccine compositions of the present disclosure may further include aconjugate of an oligosialic acid derivative as disclosed herein. Ofspecific interest is an oligosialic acid derivative conjugated to asecond molecule that is an immunomodulator. In particular embodiments,the immunomodulator is a toxin or derivative thereof, such as tetanustoxoid. Examples of tetanus toxoid conjugate vaccine compositions ofspecific interest are those selected from NPrSia-TT, DeNAc-TT, OS-TT,and TcAc-TT, as described in the experimental examples, and derivativesthereof in which a single species of the oligosialic acid derivative isprovided, for example, dimer, trimer or tetramer.

As summarized above, the disclosure provides methods of producing thealpha (2→8) and alpha (2→9) oligosialic acid derivatives disclosedherein. One feature of the methods is the use of sodium borohydride in areduction reaction to generate an alpha (2→8) or (2→9) oligosialic acidderivative bearing a reducing end enriched for de-N-acetyl residues.This method involves (i) treating an alpha (2→8) or (2→9) oligosialicacid precursor having a reducing end and a non-reducing end with sodiumborohydride under conditions for de-N-acetylating the non-reducing end,and (ii) isolating alpha (2→8) or (2→9) oligosialic acid derivativehaving one or more de-N-acetylated residues and a non-reducing end thatis resistant to degradation by exoneuraminidase. A composition ofparticular interest that is generated by this method includes anisolated alpha (2→8) or (2→9) oligosialic acid derivative having (i) adegree of polymerization of about 2-20, and (ii) a non-reducing end thatis enriched for de-N-acetyl residues and resistant to degradation byexoneuraminidase.

The sodium borohydride reduction reaction can be adjusted to generateoligosialic acid product with variable degrees of de-N-acetylation andsialic acid content. The reduction reaction is usually carried out inaqueous solution with the pH around or above pH 8. Most typically thereaction is allowed to proceed at a pH above 9, usually about 9 to 11,and most typically around 10. When the reaction is carried out around10, the de-N-acetylation appears to be preferential for the non-reducingend of the sialic acid precursor material. The pH also can be adjustedor allowed to rise over the course of the reaction. In this embodimentthe pH of the initial reaction conditions can be about 8, and the pH maybe adjusted or allowed to generally rise over the course of the reactionto about 10.

Duration of the sodium borohydride reaction and temperature are usefulvariables for adjusting the desired conditions. For example, a sialicacid precursor material such as oligosialic acid can be admixed withsodium borohydride and water and left at a suitable temperature (e.g.,ambient) and period of time (e.g., overnight) until the reaction reachesits desired endpoint (e.g., oligosialic acid derivative having areducing end that is enriched for de-N-acetyl residues and resistant todegradation by exoneuraminidase).

The reaction mixture can then be purified by standard methods (e.g.,dialysis in water and lyophilized followed by ion exchange) so as toisolate the desired material from byproduct, side reactions and thelike, for analysis, storage, formulation, further modification and/orimmediate use. For example, the amount of sialic acid and de-N-acetylsialic acid in the oligosialic acid product may be determined (e.g., byresorcinol assay, such as described in the Examples), and/or tested forits ability to inhibit binding of an antibody such as SEAM 2, SEAM 3and/or DA2 to a target antigen by inhibition ELISA, such as describedbelow, for characterization, determination of IC50, and release purposesand the like.

A feature of the sodium borohydride reaction carried out on oligosialicacid precursor material is the majority (e.g., essentially all) of theoligosialic acid derivative generated by the method contains both sialicacid and de-N-acetyl sialic acid, as opposed to only de-N-acetylatedmaterial. Another feature of the sodium borohydride reaction witholigosialic acid precursor is that the de-N-acetylation reaction mayoccur selectively at the non-reducing end. For instance, treatment ofthe reaction product with excess amounts of an exoneuraminidase does notdecrease the amount of oligosialic acid derivative, nor does it affectthe ability of the oligosialic acid derivative to inhibit SEAM 3 bindingto N-propionyl NmB polysialic acid.

Thus in another embodiment, the sodium borohydride de-N-acetylationreaction itself can be exploited to enrich for alpha (2→8) or (2→9)oligosialic acid derivative having a non-reducing end that is resistantto degradation by exoneuraminidase. This includes a specific embodimentin which the method is exploited to produce an alpha (2→8) or (2→9)oligosialic acid having a non-reducing end that is a de-N-acetylatedresidue, such as a neuraminic acid residue. The present disclosure thusprovides both alpha (2→8) or (2→9) oligosialic acid derivatives that areenriched for de-N-acetylation at a non-reducing end, as well ascompositions containing alpha (2→8) or (2→9) oligosialic acidderivatives, which compositions are enriched for alpha (2→8) or (2→9)oligosialic acid derivatives having a de-N-acetylated residue at thenon-reducing end.

In another aspect, the sodium borohydride reaction is capable ofgenerating a non-reducing end of an oligosialic acid derivative that isin a complex with boron. As sodium borohydride is a strong reducingagent, the reduction reaction may also be used to generate material inwhich the reducing end of the oligosialic acid derivative is reduced. Ineach instance, the desired material can be readily characterized by itsability to inhibit SEAM 2, SEAM 3and/or DA2 3 binding to N-propionyl NmBpolysialic acid. In other embodiments, the desired material ischaracterized by de-N-acetyl residue content. In certain embodiments,the desired material is characterized by one or more of antibodyinhibition, de-N-acetyl residue content, N-acetyl residue content,degree of polymerization, purity, complement-mediated deposition,bacterial lysis, and reduction of cell viability, such as described inthe experimental examples herein. And the compositions of the presentdisclosure may include these embodiments.

While the sodium borohydride method of the present disclosure can beoptimally applied to generate oligosialic acid derivatives, the methodmay optionally include the additional step of enriching for oligo orpolysialic acid derivative having a non-reducing end that is resistantto degradation by exoneuraminidase. The additional enriching step can becarried out by various purification methods, but advantageously bytreatment with exoneuraminidase followed by isolation of material thatis resistant to exoneuraminidase degradation, so as to simplify theprocess and improve step yield and product quality. This aspect canfacilitate further extension of the method in the generation ofde-N-acetylated product of increasingly longer chain length whileretaining a desired attribute of the end product, namely, oligosialicacid derivative bearing a non-reducing end that is enriched forde-N-acetyl residues and resistant to degradation by exoneuraminidase.

In another embodiment, the sodium borohydride method also finds use ingenerating product that is comprised as an isolated mixture of alpha(2→8) or (2→9) oligosialic acid chains. For instance, when the reactionemploys a precursor material that is polydisperse, such as an acidhydrolysis product of colominic acid, the end product is typicallypolydisperse. Thus in one embodiment, the oligosialic acid derivative isderived or obtainable from a precursor material which is itself derivedor obtainable from colominic acid or the acid hydrolysis product ofcolominic acid.

The mixtures also may have varying degrees of polymerization. Examplesinclude oligosialic acid derivatives having a degree of polymerizationranging from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 and above, including 20 to 25, 25 to 30, 35 to 50, 50 to 75, 75to 100, and 100 to 200 and above, depending on the oligosialic acidprecursor material employed in the sodium borohydride reaction. Ofspecific interest are oligo and polysialic acid derivatives (and thecompositions of the present disclosure that contain them) that include adegree of polymerization ranging from 2 to 200, with the degree ofpolymerization of oligosialic acid derivative of particular interestbeing from about 2 to 75, 2 to 70, 2 to 65, 2 to 60, 2 to 55, 2 to 50, 2to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, and more specifically 2 to20. A particular embodiment is an oligosialic acid derivative with adegree of polymerization that is a positive integer ranging from about 2to 20, as well as sub-ranges. For instance, the degree of polymerizationfor oligosialic acid derivative can be about 2 to 6, 4 to 8, 6 to 10, 8to 12, 10 to 14, 12 to 16, and about 14 to 20. In a specific example,the degree of polymerization is in a range selected from about 4 to 6, 5to 7, 6 to 9, 7 to 10, 8 to 11, 9 to 12, 10 to 13, 11 to 15, 12 to 18and 13 to 20. In other embodiments, the degree of polymerization can bewithin or overlapping with the above ranges. As noted above, the degreeof polymerization can be adjusted by selection of the precursor materialused in the sodium borohydride reaction, as well as downstreampurification by various chromatography techniques know in the art (e.g.,dialysis, high performance liquid chromatography, affinitychromatography, size exclusion chromatography, ion exchange etc.).

As noted above, the oligosialic acid material generated from the sodiumborohydride reaction will contain both sialic acid and de-N-acetylsialic acid, and the ratio of sialic acid and de-N-acetyl sialic acidcan vary depending on chain length. By way of example, a typical ratiofor short oligosialic acid derivatives ranges from roughly 3:1 to 10:1or more for the longer oligosialic acids. Thus the oligosialic acidderivatives include those having a sialic acid to de-N-acetyl sialicacid ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 and 20:1. Specific oligosialicacid derivatives of interest have a sialic acid to de-N-acetyl sialicacid ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1,13:1, 14:1 and 15:1, with those having a ration of 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1 and 10:1 being of particular interest. Thus in oneembodiment, the method generates, and thus compositions of the presentdisclosure include, a mixture of alpha (2→8) or (2→9) oligosialic acidchains that contains shorter length chains and a ratio of sialic acid tode-N-acetylated sialic acid of about 3:1. In another related embodiment,the desired product is a mixture of alpha (2→8) or (2→9) oligosialicacid chains that contains longer length chains and a ratio of sialicacid to de-N-acetylated sialic acid of about 10:1. Specific examples areshorter length chains that have a degree of polymerization ranging fromabout 2 to 6. Another example relates to longer length chains thatcomprise a degree of polymerization ranging from about 15 to 20. Yetanother example is a mixture of alpha (2→8) or (2→9) oligosialic acidchains that comprise medium length chains having a degree ofpolymerization of about 6 to 15.

In some embodiments, production of an isolated oligosialic acidderivative involves: (i) providing a first composition comprisingde-N-acetylated oligosialic acid; (ii) re-N-acylating thede-N-acetylated oligosialic acid to generate a second compositioncomprising partially re-acylated oligosialic acid having a mixture ofN-acyl and de-N-acetyl residues; and then (iii) isolating from thesecond composition oligosialic acid derivative resistant to degradationby exoneuraminidase. In this embodiment, the re-acylation may be carriedout with anhydrides, halogen functionalized, or otherwise activated acylderivatives for coupling to the free amine of the partiallyde-N-acetylated oligosialic acid material. In another method, productionof an isolated oligosialic acid derivative involves: (i) providing afirst composition comprising polysialic acid precursor; (ii) partiallyde-N-acetylating the first composition to generate a second compositioncomprising partially de-N-acylated oligosialic acid having a mixture ofN-acyl and de-N-acetyl residues, and then (ii) isolating from the secondcomposition oligosialic acid derivative resistant to degradation byexoneuraminidase.

Oligosialic acid precursors of particular interest are homopolymers,such as a homopolymer of sialic acid, for example, colominic acid, andcan be derived from natural sources or synthetic. In another embodiment,the polysialic acid precursors can be obtained from polysialic acidmaterials of Escherichia coli K1, Escherichia Coli K92, Neisseriameningitidis Serogroup B, Neisseria meningitidis Serogroup C,Haemophilus ducreyi, Campylobacter jejuni, Moraxella catarrhalis,Streptococcus algalactiae, and Paterurella multocidae. Additionalsuitable polysialic acid materials may be employed (Troy, F.,Sialobiology and the Polysialic Acid Glycotype: Occurrence, Structure,Function, Synthesis, and Glycopathology, Chpt. 4, pp. 95-133, In Biologyof Sialic Acids, Ed., Abrahman Rosenburg, Springer, 1995). Thus,depending on the precursor material selected, the N-acetyl andde-N-acetyl residues can be advantageously selected. For example, in oneembodiment, the de-N-acetyl residue is neuraminic acid. In anotherembodiment the N-acetyl residue is sialic acid. In another embodiment,the polysialic acid derivative is a homopolymer of neuraminic acid andsialic acid. In other embodiments, the N-acetyl and/or de-N-acetylneuraminic acid is O-acetylated at one or more positions, such as for apolysialic acid precursor obtained from polysialic acid of N.meningitidis Serogroup C in which C7 and C8 are O-acetylated in thenaturally occurring material. In this regard, the present disclosureprovides for control of the level of acylation of the final product, andin particular, the ability to generate oligosialic acid derivative thatcontains the desired mixture of residues.

This includes a related embodiment in which the polysialic acidprecursor is selected so as to generate oligosialic acid derivative thatcontains about 10% to 98% de-N-acetyl residues, usually about 10% toabout 60%, and in certain embodiments, about 1, 2, 3, 4 or 5 de-N-acetylresidues per oligosialic acid chain, and in specific embodiments, about1 de-N-acetyl residues per oligosialic acid chain. Thus alsocontemplated herein are polysialic acid precursor selected so as togenerate oligosialic acid derivative that contains a non-reducing endde-N-acetyl residue linked through a glycosidic bond to a residueselected from an N-acetyl residue and an N-acylated residue other thanan N-acetyl group, and where the oligosialic acid derivative issubstantially unoxidized and purified oligosaccharide having a degree ofpolymerization of about 2-10.

In a related embodiment, the polysialic acid precursor of the presentdisclosure can also be modified with various non-natural N-acyl groups.For instance, the polysialic acid precursor may re-N-acylated as notedabove, or be the product of biosynthesis of a polysialic acid in cellculture where the growth media is supplemented with a mixture ofmannosamine derivatives (e.g., N-trihaloacyl mannosamine) and acylmannosamine (e.g., N-trihaloacetyl and N-acetyl mannosame) in a desiredratio such that the precursor material expressed by the cells containsthe desired mixture of de-N-acetyl and N-acetyl residues, as well as thedesired amount of non-natural N-acyl groups.

In the re-N-acylation step, partial re-N-acylation provides forproduction of a polysialic acid derivative having fewer than 90%, fewerthan 85%, fewer than 84%, fewer than 80%, fewer than 75%, fewer than70%, fewer than 60%, or fewer than 55%, usually about 10%, about 15%,about 16%, about 20%, about 25%, about 30%, about 40%, or about 45%N-acylated residues relative to the total residues of the compound. Inthis regard, the present disclosure provides for control of the level ofacylation of the final product, so as to provide oligosialic acidderivative having a desired level of acylation. In general, reacylationis controlled or prevented by limiting the amount of acylating reagent.A particular embodiment of interest is oligosialic acid derivativehaving about about 1, 2, 3, 4 or 5 re-N-acylated residues peroligosialic acid chain, and in specific embodiments, about 1re-N-acylated residues per oligosialic acid chain.

Other approaches are possible as well, including re-N-acylation with amixture of amine protected group and acyl groups (e.g., trihaloacetyland acetyl groups) in a desired ratio such that the polysialic acidderivative contains fewer than 90%, fewer than 85%, fewer than 84%,fewer than 80%, fewer than 75%, fewer than 70%, fewer than 60%, fewerthan 55% amine protected residues, usually about 10%, about 15%, about16%, about 20%, about 25%, about 30%, about 40%, or about 45% amineprotected residues (e.g., N-trihaloacylated residues) relative to thetotal residues of the compound (where the compound generally contains atleast 10 or at least 20 residues). In this regard, the presentdisclosure provides for control of the level of acylation of the finalproduct after removal of the amine protecting group and avoidingundesirable side reactions with free amino groups, so as to provide apolysialic acid derivative having a desired level of acylation. Removalof the amine protecting groups for a free amine at the deprotectedresidue. In general, the proportion of de-N-acetyl residues iscontrolled by limiting the amount of amine protecting reagent (e.g, theamount of a trihaloacylting reagent). Here again, one embodiment ofspecific interest is the generation of oligosialic acid derivativecontaining the desired mixture of de-N-acetyl and N-acetyl residues, aswell as the desired amount of non-natural N-acyl group as noted above.

In a specific embodiment, the first composition of the method ofproduction is provided by treating a polysialic acid precursor with astrong reducing agent (e.g., sodium borohydride) in conjunction with orfollowed by a strong base (e.g., sodium hydroxide) under conditionssuitable for de-N-acetylating the precursor.

The reaction mixture can then be purified by standard methods (e.g.,dialysis in water and lyophilized followed by ion exchange) so as toisolate the desired material from byproduct, side reactions and thelike. For example, the quality of the material and amount of particularresidues in the oligosialic acid product may be determined at this point(e.g., by resorcinol assay, such as described in the Examples), and/ortested for its ability to be taken up and expressed as antigen on thesurface of a cell, such as described below, for characterization, andrelease purposes and the like.

When coupled to the isolation of oligosialic acid derivatives resistantto degradation by exoneuraminidase, the products are enriched with thedesired material and particularly well suited for increasing the antigencontent on the surface of a cell. A composition of particular interestgenerated by this method includes an isolated oligosialic acidderivative having a non-reducing end that is enriched for de-N-acetylresidues and resistant to degradation by exoneuraminidase, as well ascompositions that are enriched with mixtures of oligosialic acidderivatives having a non-reducing end enriched for de-N-acetyl residues.

For instance, the method of production step of isolating oligosialicacid derivative resistant to degradation by exoneuraminidase from thesecond composition typically involves exposing the partially re-acylatedoligosialic acid to exoneuraminidase, and then purifying the desiredoligosialic acid derivative. Exoneuraminidase of particular interest isan exosialidase from Arthrobacter ureafaciens (SIALIDASE A™, Prozyme,Hayward, Calif.). In this aspect, exoneuraminidase (exosialidase) cannotdegrade polysialic acid that terminates on the non-reducing end with ade-N-acetyl sialic acid residue (i.e., neuraminic acid) or one that isotherwise chemically blocked. Therefore, digestion of a preparation ofoligosialic acid derivative that contains de-N-acetyl residues locatedthroughout the polymer with an exoneuraminidase will result indegradation of the polysialic acid except when the exoneuraminidaseencounters a de-N-acetyl residue. At that point, no further degradationof the polymer will occur. Also, the oligosialic acid molecules that arenot degraded are likely to have a de-N-acetyl sialic acid residue at thenon-reducing end. Alternatively, the desired material can be isolated bystandard purification of derivative under conditions that select for aterminal non-reducing end that is blocked from degradation byexoneuraminidase, such as a terminal neuraminic acid residue and thelike.

Thus, in certain embodiments, the method of production can be used todirectly produce a desired polysialic acid derivative resistant todegradation by exoneuraminidase from precursor material appropriate forthis purpose. This method involves: (i) treating a first compositioncomprising oligosialic acid derivative having a mixture of N-acetyl andde-N-acetyl residues with exoneuraminidase; and (ii) isolating from thefirst composition oligosialic acid derivative resistant to degradationby the exoneuraminidase. This method is particularly suited when theprecursor material is appropriately selected and/or prepared to containa mixture of N-acetyl and de-N-acetyl residues, and then the desiredproduct purified and isolated away from the degradation products so asto avoid unwanted side reactions such as re-acetylation, aldehyde andketone side reactions, unwanted cross linking, as well as a wide rangeof other unwanted contaminants such as monomer and intermediatessusceptible to exoneuraminidase degradation, or that otherwise alter thedesired properties of the material.

In another specific embodiment, compositions of the present disclosurecan be produced by (i) providing a solution comprising a mixture ofoligosialic acid derivatives each having: a different degree ofpolymerization, a different mixture of N-acyl residues and de-N-acetylresidues, and a non-reducing end N-acetyl sialic acid residue; (ii)subjecting the solution to ion exchange chromatography to generatefractions; and (iii) isolating from one or more of the fractionsoligosialic acid derivative having a defined degree of polymerizationand a non-reducing end de-N-acetyl residue resistant to degradation byexoneuraminidase. In certain aspects, the mixture of oligosialic acidderivatives further includes oligosialic acid molecules having anon-reducing end N-acetyl group. In some embodiments, the oligosialicacid derivative having a defined degree of polymerization is isolated inan individual fraction, or a pool of fractions formed by poolingselected fractions containing a polysialic acid derivative having adesired activity of interest.

In particular embodiments, ion exchange chromatography is carried out ata pH of between about 6.5 and about 10.0. In a specific embodiment, theion exchange chromatography is anion exchange chromatography. In someembodiments, the anion exchange chromatography is high pH anion-exchangechromatography (HPAC). In certain embodiments, the anion exchangechromatography utilizes DEAE, TMAE, QAE, or PEI. In other embodiments,the anion exchange chromatography utilizes Toyopearl Super Q 650M,MonoQ, Source Q or Fractogel TMAE. A particular ion exchangechromatography procedure of interest employs a resin such as QSepharose™ Fast Flow (strong anion), SP Sepharose™ Fast Flow (strongcation), CM Sepharose™ Fast Flow (weak cation), DEAE Sepharose™ FastFlow (weak anion), and ANX Sepharose™ 4 Fast Flow (high sub) (weakanion) (e.g., available from GE Healthcare Bio-Sciences Corp.,Piscataway, N.J.). Of specific interest are strong anion exchangers,such as Q Sepharose™ Fast Flow. Sample/loading buffer and elution systemfor such ion exchange columns and systems are generally selected forresolving the isolation of a particular compound of interest.

An example of a general buffer system for a Q Sepharose™ Fast Flow anionexchange resin is a sample/loading buffer system of 20 mM Bis-Trisbuffer, pH 8, and an elution buffer system composed of a 0M to 0.2Mgradient of sodium chloride in 20 mM Bis-Tris buffer, which can beeluted at different flow rates depending on column dimensions and thelike. The ion exchange fractions containing a de-N-acetyl and N-acetylsialic acid material of interest can be analyzed with great sensitivityby high pH anion-exchange chromatography with pulsed amperometricdetection (HPAC-PAD)(e.g., Townsend, R. R. (1995) Analysis ofglycoconjugates using high-pH anion-exchange chromatography. J.Chromatog. Library 58, 181-209; and Manzi et al., (1990) HPLC of sialicacids on a pellicular resin anion exchange column with pulsedamperometry. Anal. Biochem. 188, 20-32). The isolated material may bepurified further by one or more orthogonal chromatography techniquessuch as gel permeation, size exclusion, RP-HPLC and the like. Ifdesired, the isolated oligosialic acid material can be subjected to oneor more of further preparatory steps, such dialysis, lyophilization,crystallization, formulation and the like.

The ion exchange and purification method described above can be carriedout on a mixture of oligosialic acid derivative that is produced bytreating a first composition comprising oligosialic acid derivativehaving a mixture of N-acetyl and de-N-acetyl residues withexoneuraminidase. The method may also be carried out on a mixture ofre-acetylated oligosialic acid derivatives, such as produced byre-acylating a first composition comprising de-N-acetylated oligosialicacid to generate a second composition, the second composition comprisingpartially re-acylated oligosialic acid having: a mixture of N-acyl andde-N-acetyl residues, and which is resistant to degradation byexoneuraminidase.

In a particular embodiment of interest, the ion exchange andpurification method described above is applied in the production andpurification of isolated oligosialic acid derivative that issubstantially unoxidized and defined so as to have few side products inthe initial material subjected to ion exchange purification. Forinstance, unwanted oxidation of oligosialic acid generates multipleoverlapping degradation and side reaction products that can be difficultto resolve and separate from the desired material by ion exchangechromatography. As such, “substantially unoxidized” is intended meanthat the oligosialic acid derivative, excepting normal isomer ortautomer equilibriums, contains less than about 20%, less than about15%, less than about 10%, less than about 5% oxidized sacchardideresidues, and usually about 80%, about 85%, about 90%, about 95% orgreater unoxidized sacchardide residues. Of specific interest is a totalchemical synthesis method that generates an initial product containingfew side reaction products, and facilitates the purification of smalleroligosialic acid derivatives of defined length and composition.

In certain embodiments, the substantially unoxidized and definedoligosialic acid derivative is produced by time-controlledde-N-acetylation and/or non-oxidizing acid hydrolysis of a oligosialicacid precursor material of interest. A featured aspect is a chemicalsynthesis method for the production of a substantially unoxidized anddefined oligosialic acid derivative, where the method involves either(i) non-oxidizing acid hydrolysis of partially de-N-acetylatedoligosialic acid prepared by reduced time-controlled alkalinehydrolysis, or (ii) partial de-N-acetylation of oligosialic acid byreduced time-controlled alkaline hydrolysis followed by non-oxidizingacid hydrolysis.

Partial de-N-acetylation of oligosialic acid by time-controlled alkalinehydrolysis involves (i) treating a oligosialic acid precursor with astrong reducing agent in a strong base under conditions suitable forpartially de-N-acetylating the precursor, where the treating is for aperiod of time effective to generate a minimally degraded product ofpartially de-N-acetylated oligosialic acid. In certain embodiments, theperiod of time for treatment is about 1 hour or less, generally rangingfrom about 5-55 minutes in one minute increments, such as ranging fromabout 10-50 minutes, 15-45 minutes, 20-40 minutes, and usually about 40minutes. Thus, the reaction time can be selected to provide forminimally degraded product, generating desired fractions of partiallyde-N-acetylated polysialic acid separatable by ion exchangechromatography. An example of a suitable strong reducing agent for thisprocedure is sodium borohydride, sodium cyanogen borohydride and thelike (i.e., reagents that easily lose (or donate) electrons, such as inapproximate increasing order of strength: sodium cyanogenborohydride˜sodium triacetoxyborohydride, sodium borohydride, lithiumtri-sec-butylborohydride, and lithium aluminum hydride). An example of asuitable strong base is sodium hydroxide (i.e., a base which hydrolyzescompletely, raising the pH of the solution towards 14, and thus a basehaving a pKa of more than about 13, such as in approximate increasingorder of strength: potassium hydroxide, barium hydroxide, cesiumhydroxide, sodium hydroxide, strontium hydroxide, calcium hydroxide,lithium hydroxide, and rubidium hydroxide). The reaction may also beaided by selecting an appropriate temperature, usually ranging fromabout 70° C.-120° C., about 80° C.-110° C., and more typically about 90°C.-100° C. As such, alkaline de-N-acetylation can be carried out fordifferent reaction times to generate de-N-acetyl polysialic acidcontaining defined amounts of de-N-acetyl sialic acid residuesthroughout the polymer precursor, and to generate discrete fractionswith minimal overlapping degradation products. In addition, thetime-controlled partial alkaline de-N-acetylation procedure can generateoligosialic acid derivative containing desired amounts of de-N-acetylresidues, for example, about 25%-60% de-N-acetyl residues.

Non-oxidizing acid hydrolysis can be carried out to increase thefraction of chains containing de-N-acetyl sialic acid at thenon-reducing end, since the glycosidic bond at the reducing end of ade-N-acetyl sialic acid residue in oligosialic acid is resistant tohydrolysis while the bond at the non-reducing end of the residue is not.In addition, performing the acid hydrolysis reaction under suchnon-oxidizing conditions minimizes oxidative damage to thepolysaccharide that can occur in the presence of strong acid or highconcentrations (10%) of acetic acid. Furthermore, non-oxidizing acidhydrolysis facilitates the production of smaller oligosialic acid (oroligosaccharide) derivatives enriched for de-N-acetyl sialic acidresidues at the non-reducing end. This aspect of the present disclosureinvolves (i) exposing a polysialic acid precursor or a partiallyde-N-acetylated polysialic acid under acidic conditions capable ofselectively hydrolyzing a glycosidic bond of the polysialic acid, wherethe acidic conditions include a buffer solution in which dissolvedgasses have been evacuated (e.g., by alternately freezing and thawingthe solution under vacuum). Anti-oxidants and free radical scavengersmay also be added to the reaction mixture to further reduce theoxidizing environment of the reaction solution. In addition to thenon-oxidizing conditions, the acidic buffer system generally includesthose suitable for acid-based polysialic acid hydrolysis reactions, forexample, 0.1 M sodium acetate buffer, pH 5.5. Additional examples ofacidic conditions include hydrochloric acid (e.g., 20 mM HCl) andtrifluroacetic acid (e.g., 0.1 M TFA). The non-oxidizing acid hydrolysisreaction can be carried out for different periods of time, for a givenend use, which is usually about 1-30 hours, 5-25 hours, 10-20 hours, andgenerally about 15-18 hrs. The temperature of the reaction may also beadjusted to aid control of the reaction. Examples of suitable atemperature range is about 25° C. or greater, such as a temperature ofabout 40° C. to 90° C., usually about 50° C. to 70° C. As such, thenon-oxidizing acid hydrolysis method is well suited for generatingshorter length oligosialic acid derivatives having a non-reducing endde-N-acetyl residue and a desired degree of polymerization, includingfor example, products with a defined degree of polymerization of about2-20, usually of about 2-10.

Hence the products produced by the methods include certain features togenerate product that is substantially free of contaminants, and thusenriched for the desired derivative relative to non-enriched controls.This includes oligosialic acid derivatives that have an increase in theproportion of a desirable property or element. For example, isolation ofa desired oligosialic acid derivative is where the oligosialic acid ofinterest represents the majority of the desired material (e.g., morethan 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more up to100%). This of course includes mixtures of oligosialic acid derivativeshaving variable chain lengths, provided that the majority of chains eachindividually contain a mixture of de-N-acetyl and N-acyl residues andthat is resistant to degradation to exoneuraminidase, as well asmixtures with these features and the additional feature of having anon-reducing end that is enriched for de-N-acetyl residues, includingfor instance a de-N-acetylated residue at the non-reducing terminal end(i.e., a non-reducing end de-N-acetyl sialic acid residue).

Again, depending of the specific approach, oligosialic acid derivativecan be produced to have various beneficial structural and relatedfunctional properties, such as a non-reducing end having one or morede-N-acetyl residues, a terminal de-N-acetyl residue and the like. Asnoted above, a de-N-acetyl residue of specific interest is neuraminicacid, and thus the terminus of the non-reducing end can be neuraminicacid. As also noted above, the methods can be exploited to produceoligosialic acid derivative in which the non-reducing end is enrichedwith de-N-acetyl residues, as well as homopolymers of neuraminic andsialic acid and the like.

The production methods of present disclosure also feature additionalproducts that can be produced or derived from the methods. Inparticular, the methods may further include the step of conjugating asecond molecule. In this aspect, the isolated alpha (2→8) or (2→9)oligosialic acid derivative is conjugated to a second molecule, such asa protecting group, amino acid, peptide, polypeptide, lipid,carbohydrate, nucleic acid, detectable label and the like.

An advantage of oligosialic acid derivatives that are conjugated toanother molecule includes the ability to retain the desired activity,while exploiting properties of the second molecule of the conjugate toimpart an additional desired characteristic. For example, theoligosialic acid derivatives can be conjugated to a second molecule suchas a peptide, polypeptide, lipid, carbohydrate and the like that aids insolubility, storage or other handling properties, cell permeability,half-life, controls release and/or distribution such as by targeting aparticular cell (e.g., neurons, leucocytes etc.) or cellular location(e.g., lysosome, endosome, mitochondria etc.), tissue or other bodilylocation (e.g., blood, neural tissue, particular organs etc.). Otherexamples include the conjugation of a dye, fluorophore or otherdetectable labels or reporter molecules for assays, tracking and thelike. More specifically, the oligosialic acid derivatives describedherein can be conjugated to a second molecule such as a peptide,polypeptide, dye, fluorophore, nucleic acid, carbohydrate, lipid and thelike (e.g., at either the reducing or non-reducing end), such as theattachment of a lipid moiety, including N-fatty acyl groups such asN-lauroyl, N-oleoyl, fatty amines such as dodecyl amine, oleoyl amine,and the like (e.g., see U.S. Pat. No. 6,638,513)).

In a specific embodiment of the present disclosure, the conjugatemodifies cellular uptake relative to unconjugated material. In a relatedembodiment, the oligosialic acid derivative conjugate increases cellularuptake relative to unconjugated material. In other embodiments, theconjugate decreases cellular uptake relative to unconjugated material.In this aspect, the efficiency of cellular uptake can be increased ordecreased by linking to peptides or proteins that facilitateendocytosis. For example, a given oligosialic acid derivative can belinked to a ligand for a target receptor or large molecule that is moreeasily engulfed by endocytotic mechanisms, such as an antibody. Theantibody or other ligand can then be internalized by endocytosis and thepayload released by acid hydrolysis or enzymatic activity when theendocytotic vesicle fuses with lysosomes. As such, the conjugate may beone that increases endocytosis relative to unconjugated oligosialic acidderivative. To decrease cellular uptake, the conjugate can include aligand that retains the oligosialic acid derivative on the surface of acell, which can be useful as a control for cellular uptake, or in someinstances decrease uptake in one cell type while increasing it inothers.

Other features of the conjugates can include one where the conjugatereduces toxicity relative to unconjugated oligosialic acid derivative.In further embodiments, the conjugate targets a cancer cell relative tounconjugated material. Additional examples include a conjugate theoligosialic acid derivative with one or more molecules that complement,potentiate, enhance or can otherwise operate synergistically inconnection with the oligosialic acid derivative. For instance, theoligosialic acid derivative can optionally have attached an anti-cancerdrug for delivery to a site of a cancer cell to further facilitate tumorkilling or clearance, e.g., an anti-proliferation moiety (e.g., VEGFantagonist, e.g., an anti-VEGF antibody), a toxin (e.g., an anti-cancertoxin, e.g., ricin, Pseudomonas exotoxin A, and the like), radionuclide(e.g. 90Y, 131I, 177L, 10B for boron neutron capture, and the like),anti-cancer drugs (e.g. doxorubicin, calicheamicin, maytansinoid DM1,auristatin caupecitabine, 5-fluorouricil, leucovorin, irinotercan, andthe like), and/or can optionally be modified to provide for improvedpharmacokinetic profile (e.g., by PEGylation, hyperglycosylation, andthe like).

The oligosialic acid and conjugate compositions also include alpha (2→8)or (2→9) oligosialic acid derivatives having one or more re-N-acylatedresidues. For example, a re-N-acylated residue of specific interestcomprises an amino protecting group. Exemplary amino protecting groupsfor use include, but are not necessarily limited to, carbamates, amides,N-alkyl and N-aryl amines, imine derivatives, enamine derivatives,N-sulfonyls, and the like. Further exemplary amine protecting groupsinclude, but are not necessarily limited to: acyl types such as formyl,trifluoroacetyl, phthalyl, and p-toluenesulfonyl; aromatic carbamatetypes such as benzyloxycarbonyl (Cbz) and substitutedbenzyloxy-carbonyls, 1-(p-biphenyl)-1-methylethoxy-carbonyl, and9-fluorenylmethyloxycarbonyl (Fmoc); aliphatic carbamate types such astert-butyloxycarbonyl (tBoc), ethoxycarbonyl,diisopropylmethoxycarbonyl, and allyloxycarbonyl; cyclic alkyl carbamatetypes such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; alkyltypes such as triphenylmethyl and benzyl; trialkylsilane such astrimethylsilane; and thiol containing types such as phenylthiocarbonyland dithiasuccinoyl. Amine protecting groups and protected amine groupsare described in, e.g., C. B. Reese and E. Haslam, “Protective Groups inOrganic Chemistry,” J. G. W. McOmie, Ed., Plenum Press, New York, N.Y.,1973, Chapters 3 and 4, respectively, and T. W. Greene and P. G. M.Wuts, “Protective Groups in Organic Synthesis,” Second Edition, JohnWiley and Sons, New York, N.Y., 1991, Chapters 2 and 3. In someembodiments, the re-acylated residues include an N-substituted groupsuch as acryl, methacryl, haloacetyl, propionyl, methanesulfonyl, di andtri-halo acetyl and the like. Re-acylated residues having anN-substituted group may therefore include a trihaloacyl group, such astrihaloacetyl and trihalopropionyl groups (e.g., trichloroacetyl,trifluoroacetyl, trichloropriopionyl, trifluoropriopionyl), and thelike. Re-acylated residues having an N-substituted trihaloacetyl orproprionyl group are of specific interest, with trichloroacetyl groupsbeing of particular interest.

A particular embodiment of interest is where the second molecule is animmunomodulator. By “immunomodulator” is intended a molecule thatdirectly or indirectly modifies an immune response. A specific class ofimmunomodulators includes those that stimulate or aid in the stimulationof an immunological response. Examples include antigens and antigencarriers such as a toxin or derivative thereof, including tetanustoxoid. Another embodiment includes an oligosialic acid derivativecomposition that contains one or more immunogenic excipients; in thisembodiment, the oligosialic acid derivative can be conjugated or not.Nevertheless, a particular feature of the oligosialic acid derivative isthat it is capable of inhibiting SEAM 2, SEAM 3 and/or DA2 binding toN-propionyl NmB polysialic acid.

Other examples include pharmaceutical compositions for use as vaccines,anti-cancer therapeutics that contain an oligosialic acid derivative ofthe present disclosure, as well as use of the derivatives for thegeneration of antibodies and the like. Compositions of particularinterest include an antibody specific for an alpha (2→8) or (2→9)oligosialic acid derivative produced according to the sodium borohydridemethod discussed above. The antibody is capable of complement mediatedbacteriolysis and opsonophagocytosis of Neisseria meningitidis group B(NmB) bacteria. Of particular interest are antibodies capable of bindingneuraminic acid-containing antigens expressed by dividing ornon-dividing Jurkat T-cell leukemia cells. An advantage of bindingnon-dividing and dividing cells is that the antibody can bind tonon-dividing Jurkat T-cell leukemia cells better than SEAM 3. Theantibodies can be polyclonal or monoclonal, and be of an animal (e.g.,mouse), human or humanized, as well as fragments thereof. The antibodiesof the present disclosure may also be conjugated, such as describedabove for the oligosialic acid derivatives.

Selected monoclonal antibodies of interest can be expanded in vitro,using routine tissue culture methods, or in vivo, using mammaliansubjects. For example, pristane-primed mice can be inoculated with logphase hybridoma cells in PBS for ascites production. Ascites fluid canbe stored at −70° C. prior to further purification. A particularembodiment of interest is an isolated antibody specific for an alpha(2→8) or (2→9) oligosialic acid derivative that comprises a non-reducingend enriched for one or more de-N-acetylated residues and is resistantto degradation by exoneuraminidase. Examples include an antibody iscapable of complement mediated bacteriolysis and opsonophagocytosis ofNeisseria meningitidis group B (NmB) and group C (NmC) bacteria.Additional examples in clued an isolated antibody capable of bindingneuraminic acid-containing antigens expressed by dividing ornon-dividing Jurkat T-cell leukemia cells. In certain embodiments, theisolated antibody binds the non-dividing Jurkat T-cell leukemia cellsbetter than SEAM 3. In certain embodiments, the isolated antibody ismouse. In a specific embodiment, the isoalted antibody is specific fornon-reducing end de-N-acetyl sialic acid residue. A featured aspect is amonoclonal antibody having a CDR polypeptide sequence selected from aCDR polypeptide sequence depicted in FIG. 19 or 20. A specificembodiment is monoclonal antibody DA2 having a light and heavy chaincomplementarity determining region (CDR) polypeptide sequence asdepicted in FIGS. 19 and 20.

In other embodiments, the monoclonal antibody is a humanized monoclonalantibody. For instance, chimeric antibodies may also be provided,especially if the antibodies are to be used in preventive or therapeuticpharmaceutical preparations. Chimeric antibodies composed of human andnon-human amino acid sequences may be formed from the mouse monoclonalantibody molecules to reduce their immunogenicity in humans by standardtechniques known in the art.

Antibody fragments (e.g., such as F(ab′)2, FV, and sFv molecules) mayalso be provided that are capable of exhibiting immunological bindingproperties of the parent monoclonal antibody molecule can be producedusing known techniques as well. For instance, a phage-display system canbe used to expand the monoclonal antibody molecule populations in vitro.Once generated, the phage display library can be used to improve theimmunological binding affinity of the Fab molecules using knowntechniques. The coding sequences for the heavy and light chain portionsof the Fab molecules selected from the phage display library can beisolated or synthesized, and cloned into any suitable vector forexpression (e.g, bacterial, yeast, insect, amphibian and mammalianvector systems).

Compositions of specific interest, including pharmaceuticalformulations, include those comprising an aggregate of a alpha (2→8) or(2→9) oligosialic acid derivative, including an aggregate of individualor a mixture of different alpha (2→8) or (2→9) oligosialic acidderivatives, and capable of being taken up by cells and expressed on thecell surface better than the corresponding non-aggregated derivative,for example, as gauged by the amount of the alpha (2→8) or (2→9)oligosialic acid derivative present on the cell surface relative to theappropriate control.

The aggregates can be molecular aggregates or microscopic aggregates.Aggregates of specific interest are particles, such as a microscopicparticle. This includes an aggregate that is capable of being morereadily taken up by the cell and expressed on the cell surface comparedto the corresponding non-aggregated derivative. By “correspondingnon-aggregated derivative” is intended the same derivative found in theaggregate in reference.

Another embodiment is a method of producing a composition comprising anaggregate of one or more an alpha (2→8) or (2→9) oligosialic acidderivatives, as well as the compositions produced by the methods. Thismethod involves exposing an alpha (2→8) or (2→9) oligosialic acidderivative to an aggregating condition so as to form an aggregate. Thusthe methods of production described above may further include the stepof forming an aggregate of the isolated alpha (2→8) or (2→9) oligosialicacid derivative. Examples of the aggregating conditions include heating,addition of an excipient that facilitates aggregation, and the like.

By “aggregate” is intended a particle comprising an aggregated complexof individual monomers of a molecule and having a combined molecularweight that is a multiple of the molecular weight of an individualmonomer of the complex. For example, an aggregate of one or moremonomers of an alpha (2→8) or (2→9) oligosialic acid derivative includean aggregate complex having a particle molecular weight that is 10× ormore of the molecular weight of an individual monomer in the aggregatedmonomer complex. This includes an aggregate having a particle with amolecular weight of greater than about 50,000, to greater than about250,000 Daltons, to greater than 500,000 Daltons, to greater than750,000 Daltons, to greater than 1,000,000 Daltons up to a particlehaving a uniform particle size that is readily visible by lightmicroscopy, e.g., under a standard low magnification light microscope(e.g., 40× magnification).

Thus, the aggregate can be a molecular or microscopic particle. Formicroscopic particles, the optimal aggregate can be selected by varyingthe mean aggregate diameter, e.g., 1 um to 20 μm, and usually about orsmaller than the diameter of a cell targeted for exposure and uptake ofthe material of interest, e.g., cells are usually approximately 1-20 μmin diameter. For non-visible molecular particles, as well as themicroscopic particles, the desired aggregate can be selected bymeasuring uptake and internalized by cells. In each instance, theaggregate of the alpha (2→8) or (2→9) oligosialic acid derivative iscapable of being taken up and internalized by cells better thannon-aggregated derivative relative to each other, a control, and/orboth.

As noted above, the aggregate can be formed by admixing a non-aggregatedforms of one or more alpha (2→8) or (2→9) oligosialic acid derivativesunder aggregating conditions, by partial degradation or partialhydrolysis of a alpha (2→8) or (2→9) oligosialic acid derivative underaggregating conditions, forming an aggregate of the alpha (2→8) or (2→9)oligosialic acid derivative with an aggregating excipient, or acombination thereof. By “aggregating condition” is intendedchemical-physical conditions that cause an otherwise soluble material toform an aggregated substance in solution. For instance, a alpha (2→8) or(2→9) oligosialic acid derivative can be heated (e.g., 30° C.-70° C.)for an appropriate period of time (e.g., 1 hr to overnight) so as toform an aggregate. Typically, the temperature and duration of exposureare selected to reduce or inhibit microbial growth (e.g., reduce thepotential for contamination) while not destroying the desired activityof the aggregate.

In another embodiment, the alpha (2→8) or (2→9) oligosialic acidderivative comprises a non-reducing end that is a de-N-acetyl residue,such as neuraminic acid, and the aggregate is formed by exposing thederivative to aggregating conditions. The sodium borohydride methoddescribed above and/or treatment with exoneuraminidase enriches fornon-reducing end de-N-acetyl residues which aggregate when heatedforming particles that are readily taken up by cells. This also appliesto other alpha (2→8) or (2→9) oligosialic of sialic acid, includingnon-derivatized alpha (2→8) or (2→9) oligosialic acid as well asderivatized alpha (2→8) or (2→9) oligosialic acid.

Thus the present disclosure also provides a method of producing anaggregate of an alpha (2→8) or (2→9) oligosialic acid or alpha (2→8) or(2→9) oligosialic acid derivative. This method involves exposing analpha (2→8) or (2→9) oligosialic acid or an alpha (2→8) or (2→9)oligosialic acid derivative having a non-reducing end that is resistantto degradation by exoneuraminidase to aggregating conditions, andisolating the aggregate.

In another embodiment, the aggregate of an alpha (2→8) or (2→9)oligosialic acid derivative is formed by the addition of one or moreexcipients capable of facilitating aggregation of the derivative. Ofparticular interest are substances capable of facilitating aggregationsuch as aluminum hydroxide.

Accordingly, the present disclosure further provides various methods foruse of the compositions of the disclosed herein. One feature of themethods is that the oligosialic acid derivatives of the presentdisclosure find particular use in eliciting antibodies that can beuseful in inhibiting the growth of cancerous cells in a subject. Thismethod involves administering to the subject an effective amount of apharmaceutically acceptable formulation that comprises an antibodyspecific for an alpha (2→8) or (2→9) oligosialic acid derivative bearinga reducing end enriched for de-N-acetyl residues and resistant todegradation by exoneuraminidase. In this embodiment, the administeringfacilitates reduction in viability of cancerous cells exposed to theantibody.

Another embodiment is a method of eliciting antibodies to a cancerouscell in a subject that bears a de-N-acetylated sialic acid (deNAc SA)epitope. This method involves administering to a subject an immunogeniccomposition comprising an isolated alpha (2→8) or (2→9) oligosialic acidderivative bearing a non-reducing end enriched for de-N-acetyl residuesand resistant to degradation by exoneuraminidase, where theadministering is effective to elicit production of an antibody thatspecifically binds to a deNAc SA epitope of the cancerous cell.

Another embodiment is a method of eliciting antibodies to bacteria thatbear a de-N-acetylated sialic acid (deNAc SA) epitope, such as thosefound on polysaccharide capsules of Neisseria (e.g., N. meningitidis,particularly N. meningitidis Groups B and C) and E. coli K1. This methodinvolves administering to a subject an immunogenic compositioncomprising an isolated alpha (2→8) or (2→9) oligosialic acid derivativebearing a reducing end enriched for de-N-acetyl residues and resistantto degradation by exoneuraminidase, where the administering is effectiveto elicit production of an antibody that specifically binds to a deNAcSA epitope of a bacteria.

By a “deNAc SA epitope” is intended a molecule that has (i) maximalcross-reactivity with an antibody against polysialic acid in which oneor more residues is a de-N-acetyl neuraminic acid residue, and (ii) hasminimal to no cross-reactivity with an antibody against normalpolysialic acid, especially as presented on a non-cancerous mammalian,e.g., human, cell surface. Thus in certain embodiments the minimal deNAcSA epitope is a disaccharide of sialic acid residues in which one orboth residues contain a free amine at the C5 amino position; when one ofthe two residues is de-N-acetylated, the second residue contains anN-acetyl group (but, in some embodiments, not an N-propionyl group). Thedisaccharide unit defining this minimal epitope may be at the reducingend, the non-reducing end, or within a polymer of sialic acid residues(e.g., within a polysaccharide). De-N-acetylated residues in the contextof polysialic acid (PSA) containing N-acylated residues are immunogenicand elicit antibodies that are reactive with the deNAc SA epitope, butare minimally reactive or not detectably reactive with human PSAantigens. For example, the de-N-acetylated NmB polysaccharide epitopewas identified using a murine anti-N-propionyl Neisseria meningitidisgroup B (N-Pr NmB) polysaccharide mAb (monoclonal antibodies), SEAM 3,described in Granoff et al., 1998, J Immunol 160:5028 (anti-N-Pr NmB PSmAbs); U.S. Pat. No. 6,048,527 (anti-NmB antibodies); and U.S. Pat. No.6,350,449 (anti-NmB antibodies).

In the methods of treatment of cancer, administering of the antibodyspecific for an alpha (2→8) or (2→9) oligosialic acid derivative, or animmunogenic composition that includes such derivative facilitates areduction in viability of cancerous cells exposed to the antibody and/oroligosialic acid derivative. Advantages of these methods are that theantibody generated by administration of alpha (2→8) or (2→9) oligosialicacid derivatives can be directly or indirectly cytotoxic to cancer cellscontaining a deNAc SA epitope. Thus can have the effect of retarding orotherwise arresting cell growth, and even inducing apoptosis, leading tocell death. Another advantage is that the cytotoxicity of the antibodycan be dose dependent, and thus adjustable. Specific examples ofcancerous cells amenable to treatment by the methods include melanoma,leukemia, or neuroblastoma.

In a related embodiment, the subject being treated possesses a deNAc SAepitope. The epitope can be present inside a cell or expressed on thecell surface, such as a cancer cell or a bacteria. This aspect can bebeneficial in the context of the methods of the present disclosure inthat cells expressing or presenting a deNAc SA epitope can be moreamenable to treatment with an antibody and/or oligosialic acidderivative of the present disclosure. Of course the antibody and/oroligosialic acid derivative can be administered to a subject that isnaive with respect to the deNAc SA epitope, for example, where therapyis initiated at a point where presence of the epitope is not detectable,and thus is not intended to be limiting. It is also possible to initiateantibody and/or oligosialic acid derivative therapy prior to the firstsign of disease symptoms, at the first sign of possible disease, orprior to or after diagnosis of a primary cancer and/or metastases of acancer having a detectable deNAc SA epitope (e.g., a ganglioside orother glycoconjugate that is at least partially de-N-acetylated).

Another embodiment involves screening for the deNAc SA epitope incombination with antibody and/or oligosialic acid derivative therapy. Inthis method, cells from a subject undergoing treatment, or being testedfor susceptibility to treatment, with antibody and/or oligosialic acidderivative are screened for the presence of a deNAc SA epitope. This canbe accomplished using an antibody or antibody fragment that binds to theepitope (e.g., an antibody specific for an oligosialic acid derivativeof the present disclosure, or a SEAM 3 monoclonal antibody (ATCC DepositNo. HB-12170)). Of particular interest is the monoclonal antibody DA2 oran antibody with similar activity against non-reducing end de-N-acetylsialic acid residues. As with cancer therapies in general, an advantageof this approach is the ability to select individuals with a cellularproliferation disorder or stage of disorder likely to be more responsiveto antibody and/or oligosialic acid derivative therapy compared to thosethat are not. Another advantage of targeting a subject with cellsbearing a deNAc SA epitope is that progress over the treatment coursecan be monitored, and therapy, including dosing regimens, amounts andthe like can be adjusted accordingly.

In practicing the methods, routes of administration (path by which theantibody and/or oligosialic acid derivative is brought into contact withthe body) may vary, where representative routes of administration forthe oligosialic acid derivative are described in greater detail below.In certain embodiments, the oligosialic acid derivative is administeredby infusion or by local injection. It also can be administered prior, atthe time of, or after other therapeutic interventions, such as surgicalintervention to remove cancerous cells. The antibody and/or oligosialicacid derivative can also be administered as part of a combinationtherapy, in which at least one of an immunotherapy, a cancerchemotherapy or a radiation therapy is administered to the subject (asdescribed in greater detail below).

In the methods, an effective amount of an antibody and/or oligosialicacid derivative is administered to a subject in need thereof. Inparticular, antibody and/or oligosialic acid derivatives of specificinterest are those that inhibit growth of a cancer cell in a host whenthe compounds are administered in an effective amount. The amountadministered varies depending upon the goal of the administration, thehealth and physical condition of the individual to be treated, age, thetaxonomic group of individual to be treated (e.g., human, non-humanprimate, primate, etc.), the degree of resolution desired, theformulation of the antibody and/or oligosialic acid derivativecomposition, the treating clinician's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. For example, the amount of antibody and/or oligosialicacid derivative employed to inhibit cancer cell growth is not more thanabout the amount that could otherwise be irreversibly toxic to thesubject (i.e., maximum tolerated dose). In other cases the amount isaround or even well below the toxic threshold, but still in animmunoeffective concentration range, or even as low as threshold dose.In embodiments involving use of the alpha (2→8) or (2→9) oligosialicacid derivatives to elicit an immunoprotective and/or immunotherapeuticimmune response against a cancer cell and/or a bacterial infection(e.g., Neisseria and/or E. coli K1), the amount of alpha (2→8) or (2→9)oligosialic acid derivative administered is an amount effective toelicit an immunoprotective or immunotherapeutic immune response in thesubject against a cancer cell and/or bacterial infection, where amountto effect such immune response may vary according to a variety ofsubject—specific factors, such as those exemplified above. Where thealpha (2→8) or (2→9) oligosialic acid derivative is administered toeffect an anti-deNAc SA antibody response, the antibodies elicited canprovide for specific binding of deNAc SA epitopes on a target antigenwith little or no detectable binding to host-derived polysialic acid.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the antibody and/or oligosialicacid derivative, and thus based on the disposition of the compositionwithin the subject. This includes consideration of the route ofadministration as well as dosage amount, which can be adjusted fortopical (applied directly where action is desired for mainly a localeffect), enteral (applied via digestive tract for systemic or localeffects when retained in part of the digestive tract), or parenteral(applied by routes other than the digestive tract for systemic or localeffects) applications. For instance, administration of the antibody istypically via injection and often intravenous, intramuscular,intratumoral, or a combination thereof.

Disposition of the antibody and/or oligosialic acid derivative and itscorresponding biological activity within a subject is typically gaugedagainst the fraction of antibody and/or oligosialic acid derivativepresent at a target of interest. For example, an oligosialic acidderivative once administered can accumulate as a component of polysialicacid, a glycoconjugate or other biological target that concentrates thematerial in cancer cells and cancerous tissue. Thus dosing regimens inwhich the antibody and/or oligosialic acid derivative is administered soas to accumulate in a target of interest over time can be part of astrategy to allow for lower individual doses. This can also mean that,for example, the dose of antibody that are cleared more slowly in vivocan be lowered relative to the effective concentration calculated fromin vitro assays (e.g., effective amount in vitro approximates mMconcentration, versus less than mM concentrations in vivo).

As an example, the effective amount of a dose or dosing regimen can begauged from the IC50 of a given antibody and/or oligosialic acidderivative for inhibiting binding of SEAM 2, SEAM 3 and/or DA2, such asdescribed in the SEAM 3 Inhibition Assay described herein. By “IC50” isintended the concentration of a drug required for 50% inhibition invitro. Alternatively, the effective amount can be gauged from the EC50of a given oligosialic acid derivative. By “EC50” is intended the plasmaconcentration required for obtaining 50% of a maximum effect in vivo.

In general, with respect to the antibody and/or oligosialic acidderivatives of the present disclosure, an effective amount is usuallynot more than 200× the calculated IC50. Typically, the amount of anantibody and/or oligosialic acid derivative that is administered is lessthan about 200×, less than about 150×, less then about 100× and manyembodiments less than about 75×, less than about 6×, 50×, 45×, 40×, 35×,30×, 25×, 20×, 15×, 10× and even less than about 8× or 2× than thecalculated IC50. In one embodiment, the effective amount is about 1× to50× of the calculated IC50, and sometimes about 2× to 40×, about 3× to30× or about 4× to 20× of the calculated IC50. In other embodiments, theeffective amount is the same as the calculated IC50, and in certainembodiments the effective amount is an amount that is more than thecalculated IC50.

In other embodiments, an effect amount is not more than 100× thecalculated EC50. For instance, the amount of antibody and/or oligosialicacid derivative that is administered is less than about 100×, less thanabout 50×, less than about 40×, 35×, 30×, or 25× and many embodimentsless than about 20×, less than about 15× and even less than about 10×,9×, 9×, 7×, 6×, 5×, 4×, 3×, 2× or 1× than the calculated EC50. In oneembodiment, the effective amount is about 1× to 30× of the calculatedEC50, and sometimes about 1× to 20×, or about 1× to 10× of thecalculated EC50. In other embodiments, the effective amount is the sameas the calculated EC50, and in certain embodiments the effective amountis an amount that is more than the calculated EC50.

Effective amounts can readily be determined empirically from assays,from safety and escalation and dose range trials, individualclinician-patient relationships, as well as in vitro and in vivo assayssuch as those described herein and illustrated in the Experimentalsection, below.

In a specific embodiment, the IC50 is calculated by inhibiting antibodybinding in vitro. This aspect can be carried out by assessing theability of an oligosialic acid derivative of interest to inhibit SEAM 2,SEAM 3 and/or DA2 antibody binding to dodecylamine N-propionyl NmBpolysialic acid or N-propionyl NmB polysialic acid, such as described inthe experimental examples for inhibition of SEAM 3 binding bydodecylamine N-propionyl NmB polysialic acid. In general, the procedureis carried out by standard ELISA in which the plates are coated withdodecylamine N-propionyl NmB polysialic acid or N-propionyl NmBpolysialic acid as described in the examples at a concentration of about10 μg/ml, and then processed and employed as described in theexperimental examples to determine inhibition of antibody binding andthe IC50. These antibodies and others suitable for various aspects ofthis purpose can be employed (e.g., SEAM-2 (ATCC Deposit No. CRL-12380),SEAM 3 (ATCC Deposit No. HB-12170), SEAM-18 (ATCC Deposit No. HB-12169),and SEAM-12 (ATCC Deposit No. CRL-12381).

As noted above, another feature of the methods is that the antibodyand/or oligosialic acid derivative can be administered to the subject incombination with one or more other therapies. For example, a therapy ortreatment other than administration of antibody and/or oligosialic acidderivative composition can be administered anywhere from simultaneouslyto up to 5 hours or more, e.g., 10 hours, 15 hours, 20 hours or more,prior to or after the oligosialic acid derivative. In certainembodiments, the antibody and/or oligosialic acid derivative and othertherapeutic intervention are administered or applied sequentially, e.g.,where the antibody and/or oligosialic acid derivative is administeredbefore or after another therapeutic treatment. In yet other embodiments,the antibody and/or oligosialic acid derivative and other therapy areadministered simultaneously, e.g., where the antibody and/or oligosialicacid derivative and a second therapy are administered at the same time,e.g., when the second therapy is a drug it can be administered alongwith the antibody and/or oligosialic acid derivative as two separateformulations or combined into a single composition that is administeredto the subject. Regardless of whether administered sequentially orsimultaneously, as illustrated above, the treatments are considered tobe administered together or in combination for purposes of the presentdisclosure.

Antibody and/or oligosialic acid derivatives which find use in thepresent methods and may be present in the compositions include, but arenot limited to those with appropriate specificity and antigenicity so asto elicit an antibody that can affect growth of a cancer or bacterialcell. As such, antibody and/or oligosialic acid derivative with suchspecificity aid in achieving the intended end result of modifyingcellular proliferation of a cancer cell or a bacterial cell whileminimizing unwanted side effects and toxicity in accordance with themethods. Put differently, the antibody and/or oligosialic acidderivatives employed need not be identical to those disclosed in theExamples section below, so long as the antibody and/or oligosialic acidderivatives are able to elicit an immune response against and/or inhibitgrowth of a cancerous cell or a bacterial cell. Thus, one of skill willrecognize that a number of antibody and/or oligosialic acid derivatives(described in more detail below), can be made without substantiallyaffecting the activity of the antibody and/or oligosialic acidderivatives. This includes compositions of pharmaceutically acceptablesalts (e.g., hydrochloride, sulfate salts), solvates (e.g., mixed ionicsalts, water, organics), hydrates (e.g., water). For the oligosialicacid compositions, they may be provided in prodrug forms thereof (e.g.,esters, acetyl forms), anomers (e.g., α/β mutarotation), tautomers(e.g., keto-enol tautomerism) and stereoisomers (e.g., β-D-isomer). Italso includes various alpha (2→8) or (2→9) oligosialic acid derivativecompositions that contain one or more immunogenic excipients, such as anadjuvant, carrier and the like, as well as non-immunogenic alpha (2→8)or (2→9) oligosialic acid derivative compositions that are essentiallydevoid of adjuvant or other immunogenic excipients.

Prodrugs of the oligosialic acid derivatives of the present disclosureare also contemplated. Such prodrugs are in general functionalderivatives of the compounds that are readily convertible in vivo intothe required compounds. Thus, in the methods of the present disclosure,the term “administering” encompasses administering the compoundspecifically disclosed or with a compound which may not be specificallydisclosed, but which converts to the specified compound in vivo afteradministration to the subject in need thereof. Conventional proceduresfor the selection and preparation of suitable prodrug derivatives aredescribed, e.g., in Wermuth, “Designing Prodrugs and Bioprecursors” inWermuth, ed. The Practice of Medicinal Chemistry, 2d Ed., pp. 561-586(Academic Press 2003). Prodrugs include esters that hydrolyze in vivo(e.g., in the human body) to produce a compound described herein.Suitable ester groups include, without limitation, those derived frompharmaceutically acceptable, aliphatic carboxylic acids, particularlyalkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which eachalkyl or alkenyl moiety has no more than 6 carbon atoms. Illustrativeesters include formates, acetates, propionates, butyrates, acrylates,citrates, succinates, and ethylsuccinates. O-acetylated prodrugs are ofparticular interest. For example, one or more hydroxyl groups of anoligosialic acid derivative of the present disclosure can beO-acetylated. [00176] Whether or not a given oligosialic acid derivativeor conjugate thereof is suitable for use according to the presentdisclosure can be readily determined using various assays, such as thoseemployed in the Experimental section, below. Generally, an oligosialicacid derivative is suitable for use in the methods if it elicits animmune response in a subject that facilitates inhibition of growth of atarget cell by at least about 2 to 10-fold, usually by at least about50-fold and sometimes by at least about 100-fold to 200-fold relative toa normal control cell, as determined using the cell based assays, suchas those described in the Experimental section, below. In certainembodiments, an oligosialic acid derivative is one that elicits anantibody that reduces viability of a target cell (such as a particularbacterial cell, cancer cell or cell line), arrests growth and/or inducesapoptosis of a target cell, and/or induces cell death, as observed inthe cell-based assays described in the Experimental section below whengenerating an immune response against the cell (e.g., cytotoxicity fromenhancing deNAc SA epitope of a bacterial or cancer cell, and making itmore susceptible to killing by a secondary antibody such as describedherein or SEAM 3, and/or one or more aspects of the immune system).

It will also be appreciated that once isolated, some of the smalleroligosialic acid derivatives can be characterized and made by othertechniques, including semi-synthetic as well as standard chemicalsynthesis. For instance, such oligosialic acid derivatives can beprepared conventionally by techniques known to one of skill in the art,including as described herein and in the Examples. CMP-N-acylated sialicacid analogs and sialyltransferases may also be used in a semi-syntheticapproach (e.g., Wakarchuk et al. (2008) Glycobiology 18:177).Representative references describing various synthesis approaches,intermediates, precursors, analysis, as well as the synthesis andpreparation of conjugates, diagnostics and the like, include U.S. Pat.Nos. 4,315,074; 4,395,399; 4,719,289; 4,806,473; 4,874,813; 4,925,796;5,180,674; 5,246,840; 5,262,312; 5,278,299; 5,288,637; 5,369,017;5,677,285; 5,780,603; 5,876,715; 6,040,433; 6,133,239; 6,242,583;6,271,345; 6,323,339; 6,406,894; 6,476,191; 6,538,117; 6,797,522;6,927,042; 6,953,850; 7,067,623; and 7,129,333; the disclosures of whichare herein incorporated by reference. See also, the followingreferences: “Solid Support Oligosaccharide Synthesis and CombinatorialCarbohydrate Libraries,” Peter H. Seeberger Ed, Wiley-Interscience, JohnWiley & Sons, Inc, NY, 2001; Plante et al., Science (2001)291(5508):1523; Marcaurelle et al., Glycobiology, 2002, 12(6): 69R-77R;Sears et al., Science (2001) 291:2344-2350; Bertozzi et al., ChemicalGlycobiology (2001) Science 291:2357-2364; MacCoss et al., Org. Biomol.Chem., 2003, 1:2029; and Liang et al. Science (1996) 274(5292):1520;Kayser et al J. Biol. Chem. 1992 267:16934, Keppler et al Glycobiology2001, 1 1:1R; Luchansky et al Meth. Enzymol. 2003, 362:249; Oetke et alEur. J. Biochem. 2001, 268:4553; and WO/1997/045436; the disclosures ofwhich are herein incorporated by reference.

Pharmaceutically acceptable salts of the oligosialic acid derivativescan be prepared by treating the free acid with an appropriate amount ofa pharmaceutically acceptable base. Representative pharmaceuticallyacceptable bases are ammonium hydroxide, sodium hydroxide, potassiumhydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide,ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,ferric hydroxide, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanot,2-diethylaminoethanol, lysine, arginine, histidine, and the like. Thereaction is conducted in water, alone or in combination with an inert,water-miscible organic solvent, at a temperature of from about 0° C. toabout 100° C., and can be at room temperature. The molar ratio of theoligosialic acid compounds to base used are chosen to provide the ratiodesired for any particular salts. For preparing, for example, theammonium salts of the free acid starting material, the starting materialcan be treated with approximately one equivalent of pharmaceuticallyacceptable base to yield a neutral salt. When calcium salts areprepared, approximately one-half a molar equivalent of base is used toyield a neutral salt, while for aluminum salts, approximately one-thirda molar equivalent of base will be used.

Pharmaceutical Formulations

Also provided are pharmaceutical compositions containing the antibodiesand/or oligosialic acid derivatives employed in the methods oftreatment. The term “antibody and/or oligosialic acid derivativecomposition” is used herein as a matter of convenience to refergenerically to compositions comprising an antibody and/or oligosialicacid derivative of the present disclosure, including conjugates.Antibody and/or oligosialic acid derivative compositions can comprise anantibody and/or oligosialic acid derivative, conjugate thereof, or both.Compositions useful for modifying the growth of cells, particularlybacterial and cancer cells, are contemplated by the present disclosure.This includes the compositions comprising an aggregate in particular, asthey are readily taken up by cells. Adjuvants may also be used toenhance the effectiveness of the vaccine compositions disclosed herein.

Adjuvants can be added directly to the vaccine compositions or can beadministered separately, either concurrently with or shortly after,vaccine administration. Examples of known suitable adjuvants that can beused in humans include, but are not necessarily limited to, alum,aluminum phosphate, aluminum hydroxide, MF59 (4.3% w/v squalene, 0.5%w/v Tween 80, 0.5% w/v Span 85), CpG-containing nucleic acid (where thecytosine is unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla,ISCOMS, LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG)microparticles, Quil A, interleukins, and the like. For experimentalanimals, one can use Freund's,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicantigen.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: thesubunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining MTP-PE) formulated into submicron particles using amicrofluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBITM adjuvant system (RAS), (Ribi Immunochem,Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or morebacterial cell wall components such as monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (DETOXTM); (2) saponin adjuvants, such as QS21 or STIMULON™(Cambridge Bioscience, Worcester, Mass.) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes), whichISCOMS may be devoid of additional detergent e.g. WO 00/07621; (3)Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA);(4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19,618-622; Krieg Curropin Mol Ther 2001 3:15-24; Roman et al., Nat. Med.,1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Daviset al, J. Immunol, 1998, 160, 870-876; Chu et al., J. Exp. Med, 1997,186, 1623-1631; Lipford et al, Ear. J. Immunol., 1997, 27, 2340-2344;Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg et al, Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883;Ballas et al, J. Immunol, 1996, 157, 1840-1845; Cowdery et al, J.Immunol, 1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167,72-78; Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey etal, J. Immunol., 1996, 157,2116-2122; Messina et al, J. Immunol, 1991,147, 1759-1764; Yi et al, J. Immunol, 1996, 157,4918-4925; Yi et al, J.Immunol, 1996, 157, 5394-5402; Yi et al, J. Immunol, 1998, 160,4755-4761; and Yi et al, J. Immunol, 1998, 160, 5898-5906; Internationalpatent applications WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100,WO 98/55495, WO 98/37919 and WO 98/52581] i.e. containing at least oneCG dinucleotide, where the cytosine is unmethylated; (8) apolyoxyethylene ether or a polyoxyethylene ester e.g. WO 99/52549; (9) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or estersurfactant in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO 01/21152); (10) a saponin and animmunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO00/62800); (11) an immunostimulant and a particle of met al salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO 99/11241;(13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+a sterol) e.g. WO98/57659; (14) other substances that act as immunostimulating agents toenhance the efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), and the like. Adjuvants suitable for human use are ofparticular interest where the subject is a human. Of specific interestare the inulin-based adjuvants, which can be beneficial particularly invaccines against both pathogens and cancer (Petrovsky N. (2006) Vaccine24 Suppl 2:S2-26-9.)

The antibody and/or oligosialic acid derivative compositions, e.g., inthe form of a pharmaceutically acceptable salt, can be formulated fororal, topical or parenteral administration for use in the methods, asdescribed above. In certain embodiments, e.g., where an antibody and/oroligosialic acid derivative is administered as a liquid injectable (suchas in those embodiments where they are administered intravenously ordirectly into a tissue), an antibody and/or oligosialic acid derivativeformulation is provided as a ready-to-use dosage form, or as areconstitutable storage-stable powder or liquid composed ofpharmaceutically acceptable carriers and excipients.

Methods for producing and formulating antibody and/or oligosialic acidderivatives suitable for administration to a subject (e.g., a humansubject) are well known in the art. For example, antibody and/oroligosialic acid derivatives can be provided in a pharmaceuticalcomposition comprising an effective amount of an antibody and/oroligosialic acid derivative and a pharmaceutical excipients (e.g.,saline). The pharmaceutical composition may optionally include otheradditives (e.g., buffers, stabilizers, preservatives, and the like). Aneffective amount of antibody and/or oligosialic acid derivative isgenerally an amount effective to provide for enhancing an anti-bacterialor anti-cancer response in a subject for a desired period. A therapeuticgoal (e.g., reduction in tumor load or protection against bacterialinfection or propagation) can be accomplished by single or multipledoses under varying dosing regimen.

By way of illustration, the antibody and/or oligosialic acid derivativecompositions can be admixed with conventional pharmaceuticallyacceptable carriers and excipients (i.e., vehicles) and used in the formof aqueous solutions, tablets, capsules, elixirs, suspensions, syrups,wafers, patches and the like, but usually the antibody and/oroligosialic acid derivative will be provided as an injectable. Suchpharmaceutical compositions contain, in certain embodiments, from about0.1 to about 90% by weight of the active compound, and more generallyfrom about 1 to about 30% by weight of the active compound. Thepharmaceutical compositions may contain common carriers and excipients,such as corn starch or gelatin, lactose, dextrose, sucrose,microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate,sodium chloride, and alginic acid. Disintegrators commonly used in theformulations include croscarmellose, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid. Preservatives and thelike may also be included.

A liquid composition will generally consist of a suspension or solutionof the compound or pharmaceutically acceptable salt in a suitable liquidcarrier(s), for example, ethanol, glycerine, sorbitol, non-aqueoussolvent such as polyethylene glycol, oils or water, with a suspendingagent, preservative, surfactant, wetting agent, flavoring or coloringagent. Alternatively, a liquid formulation can be prepared from areconstitutable powder.

A composition in the form of a tablet can be prepared using any suitablepharmaceutical carrier(s) routinely used for preparing solidcompositions. Examples of such carriers include magnesium stearate,starch, lactose, sucrose, microcrystalline cellulose and binders, forexample, polyvinylpyrrolidone. The tablet can also be provided with acolor film coating, or color included as part of the carrier(s). Inaddition, active compound can be formulated in a controlled releasedosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

A composition in the form of a capsule can be prepared using routineencapsulation procedures, for example, by incorporation of activecompound and excipients into a hard gelatin capsule. Alternatively, asemi-solid matrix of active compound and high molecular weightpolyethylene glycol can be prepared and filled into a hard gelatincapsule; or a solution of active compound in polyethylene glycol or asuspension in edible oil, for example, liquid paraffin or fractionatedcoconut oil can be prepared and filled into a soft gelatin capsule.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, poly-vinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose. Lubricants that canbe used include magnesium stearate or other met allic stearates, stearicacid, silicone fluid, talc, waxes, oils and colloidal silica.

Additionally, it may be desirable to add a coloring agent to make thedosage form more attractive in appearance or to help identify theproduct.

The compounds of the present disclosure and their pharmaceuticallyacceptable salts that are active when given parenterally can beformulated for intramuscular, intrathecal, or intravenousadministration.

A typical composition for intramuscular or intrathecal administrationwill be of a suspension or solution of active ingredient in an oil, forexample, arachis oil or sesame oil. A typical composition forintravenous or intrathecal administration will be a sterile isotonicaqueous solution containing, for example, active ingredient and dextroseor sodium chloride, or a mixture of dextrose and sodium chloride. Otherexamples are lactated Ringer's injection, lactated Ringer's plusdextrose injection, Normosol-M and dextrose, Isolyte E, acylatedRinger's injection, and the like. Optionally, a co-solvent, for example,polyethylene glycol, a chelating agent, for example, ethylenediaminetetracetic acid, and an anti-oxidant, for example, sodium metabisulphitemay be included in the formulation. Alternatively, the solution can befreeze dried and then reconstituted with a suitable solvent just priorto administration.

The compounds of the present disclosure and their pharmaceuticallyacceptable salts which are active on rectal administration can beformulated as suppositories. A typical suppository formulation willgenerally consist of active ingredient with a binding and/or lubricatingagent such as a gelatin or cocoa butter or other low melting vegetableor synthetic wax or fat.

The compounds of the present disclosure and their pharmaceuticallyacceptable salts which are active on topical administration can beformulated as transdermal compositions or transdermal delivery devices(“patches”). Such compositions include, for example, a backing, activecompound reservoir, a control membrane, liner and contact adhesive. Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the compounds of the present disclosure in controlledamounts. The construction and use of transdermal patches for thedelivery of pharmaceutical agents is well known in the art. See, e.g.,U.S. Pat. No. 5,023,252, herein incorporated by reference in itsentirety. Such patches may be constructed for continuous, pulsatile, oron demand delivery of pharmaceutical agents.

In certain embodiments of interest, the antibody and/or oligosialic acidderivative composition is administered as a single pharmaceuticalformulation. It also may be administered with an effective amount ofanother agent that includes other suitable compounds and carriers, andalso may be used in combination with other active agents. The presentdisclosure, therefore, also includes pharmaceutical compositionscomprising pharmaceutically acceptable excipients. The pharmaceuticallyacceptable excipients include, for example, any suitable vehicles,adjuvants, carriers or diluents, and are readily available to thepublic. The pharmaceutical compositions of the present disclosure mayfurther contain other active agents as are well known in the art.

One skilled in the art will appreciate that a variety of suitablemethods of administering a formulation of the present disclosure to asubject or host, e.g., patient, in need thereof, are available, and,although more than one route can be used to administer a particularformulation, a particular route can provide a more immediate and moreeffective reaction than another route. Pharmaceutically acceptableexcipients are also well-known to those who are skilled in the art, andare readily available. The choice of excipient will be determined inpart by the particular compound, as well as by the particular methodused to administer the composition. Accordingly, there is a wide varietyof suitable formulations of the pharmaceutical composition of thepresent disclosure. The following methods and excipients are merelyexemplary and are in no way limiting.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachetsor tablets, each containing a predetermined amount of the activeingredient, as solids or granules; (c) suspensions in an appropriateliquid; and (d) suitable emulsions. Tablet forms can include one or moreof lactose, mannitol, corn starch, potato starch, microcrystallinecellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellosesodium, talc, magnesium stearate, stearic acid, and other excipients,colorants, diluents, buffering agents, moistening agents, preservatives,flavoring agents, and pharmacologically compatible excipients. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin, or sucroseand acacia, emulsions, gels, and the like containing, in addition to theactive ingredient, such excipients as are known in the art.

The formulations of the present disclosure can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as for use in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

Formulations suitable for topical administration may be presented ascreams, gels, pastes, or foams, containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

Suppository formulations are also provided by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more alpha(2→8) or (2→9) oligosialic acid derivatives. Similarly, unit dosageforms for injection or intravenous administration may comprise the alpha(2→8) or (2→9) oligosialic acid derivative(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present disclosure calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms depend on the particular compound employed and the effectto be achieved, and the pharmacodynamics associated with each compoundin the host.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the nature of the deliveryvehicle, and the like. Suitable dosages for a given compound are readilydeterminable by those of skill in the art by a variety of means.

Optionally, the pharmaceutical composition may contain otherpharmaceutically acceptable components, such a buffers, surfactants,antioxidants, viscosity modifying agents, preservatives and the like.Each of these components is well-known in the art. See, e.g., U.S. Pat.No. 5,985,310, the disclosure of which is herein incorporated byreference.

Other components that can be suitable for use in the formulations of thepresent disclosure can be found in Remington's Pharmaceutical Sciences,Mack Pub. Co., 18th edition (June 1995). In an embodiment, the aqueouscyclodextrin solution further comprise dextrose, e.g., about 5%dextrose.

Utility: Exemplary Applications & Related Embodiments

The methods find use in a variety of applications, where in manyapplications the methods are modulating at least one cellular function,such as mediation of polysialic acid structure and inhibition ofcancerous cell growth, or are modulating an immune response, such inimmunization of a subject to elicit antibodies that bind a deNAc SAepitope such as may be borne on a cancerous or bacterial cell (e.g.,Neisseria or E. coli K1).

In the context of modulating at least one cellular function as well asin the context of eliciting anti-cancer cell antibodies, the methods andcompositions find use in treating cellular proliferation disorders.Thus, a representative therapeutic application is the treatment ofcellular proliferative disease conditions in general, e.g., cancers andrelated conditions characterized by abnormal cellular proliferationconcomitant. Such disease conditions include cancer/neoplastic diseasesand other diseases characterized by the presence of unwanted cellularproliferation, e.g., hyperplasias, and the like. As indicated, cellularproliferation disorders include those that abnormally express the deNAcSA epitope, which can be determined using anti-deNAc SA antibody orderivatives thereof.

In the context of modulating an immune response to elicit anti-bacterialantibodies, the methods and compositions find use in elicitingimmunoprotective and/or immunotherapeutic immune response againstbacteria that bear a deNAc SA, as in capsular polysaccharide of a deNAcSA epitope-bearing Neisseria (e.g., N. meningitidis, e.g., N.meningitidis Groups B and C) or E. coli K1.

Of particular interest are antibodies that have antigen bindingspecificity for the oligosialic acid derivatives described herein or theantigen binding specificity of mAb SEAM 3. Of particular interest areantibodies that specifically bind a deNAc SA epitope with little or nodetectable binding to human polysialic acid. Examples of such antibodiesinclude those having a light chain polypeptide comprising CDR1, CDR2 andCDR3 of the variable region of a SEAM 3 or DA2 light chain polypeptideand a heavy chain polypeptide comprising CDR1, CDR2, and CDR3 of thevariable region of the heavy chain polypeptide. Additional examples ofsuch antibodies include those having a light chain polypeptidecomprising CDR1, CDR2 and CDR3 of the variable region of a DA2 lightchain polypeptide and a heavy chain polypeptide comprising CDR1, CDR2,and CDR3 of the variable region of the heavy chain polypeptide. Suchantibodies include chimeric antibodies, humanized antibodies, and thelike.

By “treatment” is meant that at least an amelioration of the symptomsassociated with the condition afflicting the host is achieved, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thecondition being treated. As such, treatment also includes situationswhere the pathological condition, or at least symptoms associatedtherewith, are completely inhibited, e.g., prevented from happening, orstopped, e.g. terminated, such that the host no longer suffers from thecondition, or at least the symptoms that characterize the condition.Thus treatment includes: (i) prevention, that is, reducing the risk ofdevelopment of clinical symptoms, including causing the clinicalsymptoms not to develop, e.g., preventing disease progression to aharmful state; (ii) inhibition, that is, arresting the development orfurther development of clinical symptoms, e.g., mitigating or completelyinhibiting an active disease, e.g., so as to decrease tumor load, whichdecrease can include elimination of detectable cancerous cells, or so asto protect against disease caused by bacterial infection, whichprotection can include elimination of detectable bacterial cells; and/or(iii) relief, that is, causing the regression of clinical symptoms.

A variety of hosts are treatable according to the methods. Generallysuch hosts are “mammals” or “mammalian,” where these terms are usedbroadly to describe organisms which are within the class mammalia,including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,and monkeys). In many embodiments, the hosts will be humans. In thecontext of anti-bacterial vaccination methods, of interest are hoststhat are susceptible to disease that can be caused by infection by adeNAc SA epitope-bearing bacteria, such as Neisseria (e.g., N.meningitidis, e.g., N. meningitidis Groups B and C) or E. coli K1.

The methods find use in, among other applications, the treatment ofcellular proliferative disease conditions in which an effective amountof the antibody or oligosialic acid derivative composition isadministered to the subject in need thereof. Treatment is used broadlyas defined above, e.g., to include prevention or at least anamelioration in one or more of the symptoms of the disease, as well as acomplete cessation thereof, as well as a reversal and/or completeremoval of the disease condition, e.g., cure.

Compositions of the present disclosure can comprise a therapeuticallyeffective amount of an antibody or oligosialic acid derivativecomposition, as well as any other compatible components, as needed. By“therapeutically effective amount” is meant that the administration ofthat amount to an individual, either in a single dose, as part of aseries of the same or different antibody or oligosialic acid derivativecompositions, is effective to inhibit the growth of a cancerous cell ina subject. Such therapeutically effective amount of antibody oroligosialic acid derivative composition and its impact on cell growthincludes cooperative and/or synergistic inhibition of cell growth inconjunction with one or more other therapies (e.g., immunotherapy,chemotherapy, radiation therapy etc.) As noted below, thetherapeutically effective amount can be adjusted in connection withdosing regimen and diagnostic analysis of the subject's condition (e.g.,monitoring for the present or absence of a cell surface epitopes using aSEAM 3 antibody or antibody specific for an oligosialic acid derivative)and the like.

The amount administered to an animal, particularly a human, in thecontext of the present disclosure should be sufficient to affect aprophylactic or therapeutic response in the animal over a reasonabletime frame, and varies depending upon the goal of the administration,the health and physical condition of the individual to be treated, age,the taxonomic group of individual to be treated (e.g., human, non-humanprimate, primate, etc.), the degree of resolution desired, theformulation of the antibody or oligosialic acid derivative composition,the treating clinician's assessment of the medical situation, and otherrelevant factors. One skilled in the art will also recognize that dosagewill depend on a variety of factors including the strength of theparticular compound employed, the condition of the animal, and the bodyweight of the animal, as well as the severity of the illness and thestage of the disease. The size of the dose will also be determined bythe existence, nature, and extent of any adverse side-effects that mightaccompany the administration of a particular compound. Thus it isexpected that the amount will fall in a relatively broad range, but cannevertheless be routinely determined through various features of thesubject such as note above.

Also, suitable doses and dosage regimens can be determined bycomparisons to anticancer or immunosuppressive agents that are known toaffect the desired growth inhibitory or immunosuppressive response. Suchdosages include dosages which result in the low dose inhibition of cellgrowth, without significant side effects. In proper doses and withsuitable administration of certain compounds, the compounds of thepresent disclosure can provide for a wide range of intracellulareffects, e.g., from partial inhibition to essentially completeinhibition of cell growth. This is especially important in the contextof the present disclosure, as this differential inhibition canpotentially be used to discriminate between cancer cells and highlyproliferative non-malignant cells. Dosage treatment may be a single doseschedule or a multiple dose schedule (e.g., including ramp andmaintenance doses). As indicated, the antibody or oligosialic acidderivative composition may be administered in conjunction with otheragents, and thus doses and regiments can vary in this context as well tosuit the needs of the subject.

The compositions of the present disclosure can be provided in apharmaceutically acceptable excipient, which can be a solution such asan aqueous solution, often a saline solution, or they can be provided inpowder form. The antibody or oligosialic acid derivative compositionsmay comprise other components, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium, carbonate, and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example, sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like.

The concentration of antibody or oligosialic acid derivative of thepresent disclosure in the pharmaceutical formulations can vary from lessthan about 0.1%, usually at or at least about 2% to as much as 20% to50% or more by weight, and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected and the patient's needs. The resultingcompositions may be in the form of a solution, suspension, tablet, pill,capsule, powder, gel, cream, lotion, ointment, aerosol or the like.

The antibody or oligosialic acid derivative compositions (which may beoptionally conjugated) can be used alone or in combination with othertherapies (e.g., antibacterial agents, other anti-cancer agents, and thelike). When used in combination, the various compositions can beprovided in the same or different formulations. Where administered indifferent formulations, the compositions can be administered at the sameor different dosage regimen (e.g., by the same or different routes, atthe same or different time (e.g., on the same or different days)), andthe like). In general, administration of the antibody or oligosialicacid derivative composition can be performed serially, at the same time,or as a mixture, as described in more detail below. Administration canbe serial, with repeated doses of antibody or oligosialic acidderivative composition. Exemplary dosage regimens are described below inmore detail.

In general, administration of an antibody or oligosialic acid derivativecomposition is accomplished by any suitable route, includingadministration of the composition orally, bucally, nasally,nasopharyngeally, parenterally, enterically, gastrically, topically,transdermally, subcutaneously, intramuscularly, in tablet, solid,powdered, liquid, aerosol form, locally or systemically, with or withoutadded excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 18th ed., Mack Publishing Company,NY (1995).

It is recognized that when administered orally, antibody or oligosialicacid derivatives should be protected from digestion. This is typicallyaccomplished either by complexing the antibody or oligosialic acidderivative with a composition to render it resistant to acidic andenzymatic hydrolysis or by packaging in an appropriately resistantcarrier such as a liposome. Means of protecting a compound of interestfrom digestion are well known in the art.

In order to enhance serum half-life, antibody or oligosialic acidderivative preparations that are injected may also be encapsulated,introduced into the lumen of liposomes, prepared as a colloid, or otherconventional techniques may be employed which provide an extended serumhalf-life. A variety of methods are available for preparing liposomes,as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467(1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. Thepreparations may also be provided in controlled release or slow-releaseforms for release and administration of the antibody or oligosialic acidderivative compositions as a mixture or in serial fashion.

The compositions also can be administered to subject that is at risk ofdisease to prevent or at least partially arrest the development ofdisease and its complications. A subject is “at risk” where, forexample, the subject exhibits one or more signs or symptoms of disease,but which are insufficient for certain diagnosis and/or who has been ormay be exposed to conditions that increase the probability of disease.For example, the antibody or oligosialic acid derivative compositionscan also be administered to subject that is at risk of a cancer, has acancer, or is at risk of metastasis of a cancer having a cell surfacedeNAc SA epitope (e.g., a cell surface ganglioside that is at leastpartially de-N-acetylated).

Antibody or oligosialic acid derivative compositions are administered toa host in a manner that provides for the inhibition of growth of acancerous cell, which may include monitor cell histology, viability,biological marker profile and the like (e.g., monitoring for thepresence or absence of selective deNAc SA epitopes etc.). Antibody oroligosialic acid derivative compositions can be administered serially oroverlapping to maintain a therapeutically effective amount as believedneeded for the desired end result (e.g., inhibition of cancerous cellgrowth). Typically, each dose and the timing of its administration isgenerally provided in an amount that is tolerated by the health of thesubject, and can be based on IC50 and/or the EC50 as noted above. Thusamounts can vary widely for a given treatment.

Therapeutic response to the dose or treatment regime may be determinedby known methods (e.g. by obtaining serum from the individual before andafter the initial immunization, and demonstrating a change in theindividual's status as noted above, for example an immunoprecipitationassay, or an ELISA, or a bactericidal assay, or a Western blot, or flowcytometric assay, or the like). The dosing may include washout periodsto allow for clearance of the initial material, followed by halting orresumption of treatment. Thus dosage strategies can be modifiedaccordingly.

In one embodiment, the antibody or oligosialic acid derivativecomposition is administered at least once, usually at least twice, andin some embodiments more than twice. In a related embodiment, theantibody or oligosialic acid derivative composition is administered incombination along a dosing schedule and course in conjunction withchemotherapy. In another embodiment, the antibody or oligosialic acidderivative composition is administered in combination with a dosingschedule and course in conjunction with immunotherapy. In yet anotherembodiment, the antibody or oligosialic acid derivative composition isadministered in combination with a dosing schedule and course inconjunction with radiation therapy. Each individual dose of the antibodyor oligosialic acid derivative composition may be administered before,during or after the complementary therapy such as immunotherapy,chemotherapy, or radiation therapy. As can be appreciated, combinationtherapies using an antibody or oligosialic acid derivative compositionmay be adjusted for a given end need.

Exemplary Cancer Therapies

The antibody and oligosialic acid derivative compositions find use in avariety of cancer therapies (including cancer prevention andpost-diagnosis cancer therapy) in a mammalian subject, particularly in ahuman. Subjects having, suspected of having or at risk of developing atumor are contemplated for therapy and diagnosis described herein.Samples obtained from such subject are likewise suitable for use in themethods of the present disclosure.

More particularly, antibody and oligosialic acid derivative compositionsdescribed herein can be administered to a subject (e.g. a human patient)to, for example, facilitate reduction of viability of cancerous cells,e.g., to reduce tumor size, reduce tumor load, and/or improve theclinical outcome in patients. In particular, antibody and oligosialicacid derivative compositions can be used to disrupt the cell cycle ofthe cancer cell, and facilitate entry of the cell into apoptosis, e.g.,by inducing cancerous cells to enter the pre-GO cell cycle phase.

In certain embodiments, the antibody and oligosialic acid derivativecompositions may be advantageously used in an anti-cancer therapy,particularly where the cancerous cells present a deNAc SA epitope on anextracellularly accessible cell surface (e.g., a deNAc SA epitope on anat least partially de-N-acetylated ganglioside or other glycoconjugate).In one embodiment, the cancer is one that presents a SEAM 3-reactiveantigen and/or a DA2-reactive antigen. Cancers that present a SEAM3-reactive antigen and/or a DA2-reactive antigen can be identified bymethods known in the art. Exemplary methods of detection and diagnosisare described below.

Where the anti-cancer therapy comprises administration of an antibodyand/or oligosialic acid derivative composition, the anti-cancer therapycan be particularly directed to dividing (replicating, proliferating)cancerous cells. As shown in the Examples below, antibody raised againstoligosialic acid derivatives were particularly effective againstcancerous cells bearing the epitope specifically bound by SEAM 3 and/orDA2 antibody. For example, the level of extracellularly accessibleantigen bound by SEAM 3 is increased during cell division as compared tonon-dividing cells, and binding of SEAM 3 drives the cell towardanaphase (into pre-GO). Since most cancers are more rapidly dividingthan normal cells of the same type, cells that possess a SEAM 3-reactiveantigen are attractive for antibody and oligosialic acidderivative-based cancer therapy. Also, the antibodies identified hereinto the oligosialic acid derivatives of the present disclosure exhibitenhanced binding relative to binding by SEAM 3 to the OS-conjugatevaccine-derived antigen relative to normal PSA control, thus havingclinical benefits in addition to SEAM 3. For example, antibodiesgenerated using an oligosialic acid derivative composition made by themethods described herein such as DA2 may bind a SEAM 3 reactive antigenwith an improved binding affinity and/or binding avidity relative tonormal PSA control. In another example, antibodies generated using anoligosialic acid derivative composition made by the methods describedherein such as DA2 may bind an epitope of a SEAM 3 reactive antigen thatis different than the epitope bound by the SEAM 3 monoclonal antibodyrelative to normal PSA control. As illustrated in the examples, DA2 washighly effective in binding as well as killing cancer cells bearing aDA2-reactive antigen.

Thus the present disclosure particularly provides anti-cancer therapydirected toward cancerous cells involving administration of antibodyand/or oligosialic acid derivative compositions having an epitoperecognized by a SEAM 3 and/or DA2 mAb. Cancers particularly amenable toantibody and/or oligosialic acid derivative therapy can be identified byexamining markers of cellular proliferation (e.g., Ki-67 antigen) and/orby examining the presence/accessibility of the deNAc SA epitope bound bySEAM 3 and/or DA2 in dividing cells or by the antibodies specific forthe oligosialic acid derivatives of the present disclosure (e.g., as inan in vitro assay).

Cancers having a cell surface-accessible deNAc SA epitope include thosehaving an at least partially de-N-acetylated ganglioside and/or aprotein having a sialic acid modification that contains a deNAc SAepitope. Cancers having de-N-acetylated gangliosides have beendescribed.

The presence of de-N-acetyl sialic acid residues in normal human tissueappears to be transient and very low abundance, being found only in afew blood vessels, infiltrating mononuclear cells in the skin and colon,and at moderate levels in skin melanocytes. It is prevalent only inabnormal cells, such as melanomas, leukemias and lymphomas. Sinceexpression of high levels of deNAc SA antigens (e.g., de-N-acetylgangliosides) occurs predominantly in cancer cells, treatment withantibody and/or oligosialic acid derivative compositions can be used toinduce cytotoxicity, and can block tumor growth. In addition, antibodyand/or oligosialic acid derivative compositions can be usedtherapeutically to effect/prevent adhesion and invasion of cancer cellsin other tissues.

Exemplary cancers presenting a deNAc SA epitope include cancer cellspresenting a de-N-acetyl ganglioside containing a de-N-acetyl sialicacid residue (e.g. GM2alpha, GM1alpha, GD1beta, GM1b, GD1c, GD1alpha,GM3, GM2, GM1, GD13, GT13, GT1halpha, GD3, GD2, GD1b, GT1b, GQ1b,Gomega1halpha, GT3, GT2, GT1c, GQ1c, and GP1c). Of particular interestare gangliosides that contain two or more sialic acid residues linked byalpha 2-8 glycosidic bonds (e.g., GD1c, GT13, GD3, GD1b, GT1b, GQ1b,Gomega1halpha, GT3, GT1c, GQ1c, and GP1c) in which at least one residueis de-N-acetylated. In some embodiments, the ganglioside that containstwo or more sialic acid residues linked by alpha 2-8 glycosidic bonds isa ganglioside other than GD3 and/or other than GM3. In some embodiments,the target of the cancer is a deNAc SA epitope other than one present ona de-N-acetylated ganglioside (e.g., a de-N-acetylated residue of asialic acid-modified protein).

In one embodiment antibody and/or oligosialic acid derivativecompositions can be used to treat cancers that present a SEAM 3 and/orDA2 reactive antigen on a cell surface, including cancers that exhibitan extracellularly accessible SEAM 3 and/or DA2-reactive antigen duringcell division.

In another embodiment antibody and/or oligosialic acid derivativecompositions can be used to treat cancers that present deNAc SA epitopeon a cell surface, including cancers that exhibit an extracellularlyaccessible reactive antigen during cell rest.

It should be noted that while deNAc SA epitopes and/or SEAM 3 and/orDA2-reactive antigens may be expressed at higher levels on a cancer cellcompared to a non-cancerous cell, this is not a limitation of thetherapies disclosed herein. For example, where the cancer involves acell type that can be replenished (e.g., B cell, T cell, or other cellof hematopoietic origin, as in leukemias and lymphomas), inhibition ofnormal cell growth can be acceptable since damage to a subject bydepleting such cells can be treated (e.g., with drugs to stimulaterepopulation of normal cells, e.g., GM-CSF, EPO, and the like).

The methods relating to cancer contemplated herein include, for example,use of antibody and/or oligosialic acid derivative therapy alone or incombination with deNAc SA antigens as a anti-cancer vaccine or therapy,as well as use of antibodies generated using deNAc SA antigens inanti-cancer vaccines (e.g., by passive immunization) or therapies. Themethods are useful in the context of treating or preventing a widevariety of cancers, including carcinomas, sarcomas, leukemias, andlymphomas.

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epitheliealcarcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be amenable to therapy by a method disclosedherein include, but are not limited to, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Leukemias that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, a) chronic myeloproliferative syndromes(neoplastic disorders of multipotential hematopoietic stem cells); b)acute myelogenous leukemias (neoplastic transformation of amultipotential hematopoietic stem cell or a hematopoietic cell ofrestricted lineage potential; c) chronic lymphocytic leukemias (CLL;clonal proliferation of immunologically immature and functionallyincompetent small lymphocytes), including B-cell CLL, T-cell CLLprolymphocytic leukemia, and hairy cell leukemia; and d) acutelymphoblastic leukemias (characterized by accumulation of lymphoblasts).Lymphomas that can be treated using a method include, but are notlimited to, B-cell lymphomas (e.g., Burkitt's lymphoma); Hodgkin'slymphoma; non-Hodgkin's lymphoma, and the like.

Other cancers that can be amenable to treatment according to the methodsdisclosed herein include atypical meningioma (brain), islet cellcarcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma(intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver),clear cell carcinoma (kidney), and neurofibroma mediastinum.

Further exemplary cancers that can be amenable to treatment using amethods disclosed herein include, but are not limited to, cancers ofneuroectodermal and epithelial origin. Examples of cancers ofneuroectodermal origin include, but are not limited to, Ewings sarcoma,spinal tumors, brain tumors, supratenbrial primative neuroectodermaltumors of infancy, tubulocystic carcinoma, mucinous tubular and spindlecell carcinoma, renal tumors, mediastinum tumors, neurogliomas,neuroblastomas, and sarcomas in adolescents and young adults. Examplesof epithelial origin include, but are not limited to, small cell lungcancer, cancers of the breast, eye lens, colon, pancreas, kidney, liver,ovary, and bronchial epithelium. In some embodiments, the methods do notinclude treatment of melanoma (i.e., the cancer is other than melanoma).In other embodiments, the methods do not include treatment of lymphoma(i.e., the cancer is other than lymphoma). In certain embodiments, themethods of the present disclosure are used to treat cancer cells knownto express de-N-acetyl gangliosides include melanomas and somelymphomas. As noted above, cancers that overexpress the precursorgangliosides GM3 and GD3 are likely to also express the greatest amountof de-N-acetyl gangliosides on the cell surface.

Combinations with Other Cancer Therapies

Therapeutic administration of the antibody and/or oligosialic acidderivative compositions can include administration as a part of atherapeutic regimen that may or may not be in conjunction withadditional standard anti-cancer therapeutics, including but not limitedto immunotherapy, chemotherapeutic agents and surgery (e.g., as thosedescribed further below). In addition, therapeutic administration of theantibody and/or oligosialic acid derivative compositions can also bepost-therapeutic treatment of the subject with an anti-cancer therapy,where the anti-cancer therapy can be, for example, surgery, radiationtherapy, administration of chemotherapeutic agents, and the like. Use ofmonoclonal antibodies, particularly monoclonal antibodies that canprovide for complement-mediated killing, and/or antibody-dependentcellular cytotoxicity-mediated killing, of a target cell are ofparticular interest (e.g., treatment with an anti-deNAc SA epitopeantibody (e.g., DA2, SEAM 3 or an antibody specific for an oligosialicacid derivative of the present disclosure) after identification of aprimary tumor composed of cells expressing a deNAc SA epitope (e.g., ade-N-acetyl ganglioside)). Cancer therapy using antibody and/oroligosialic acid derivative compositions of the present disclosure incombination with immunotherapy that employs PSA antigen/anti-deNAc SAepitope antibodies is of particular interest (U.S. Ser. No. 11/645,255and PCT Application No. US2006/048850; incorporated herein byreference).

For example, the antibody and/or oligosialic acid derivativecompositions can be administered in combination with one or morechemotherapeutic agents (e.g., cyclophosphamide, doxorubicin,vincristine and prednisone (CHOP)), and/or in combination with radiationtreatment and/or in combination with surgical intervention (e.g., pre-or post-surgery to remove a tumor). Where the alpha (2→8) or (2→9)oligosialic acid derivative is used in connection with surgicalintervention, the antibody and/or oligosialic acid derivativecompositions can be administered prior to, at the time of, or aftersurgery to remove cancerous cells, and may be administered systemicallyor locally at the surgical site. The antibody and/or oligosialic acidderivative compositions alone or in combinations described above can beadministered systemically (e.g., by parenteral administration, e.g., byan intravenous route) or locally (e.g., at a local tumor site, e.g., byintratumoral administration (e.g., into a solid tumor, into an involvedlymph node in a lymphoma or leukemia), administration into a bloodvessel supplying a solid tumor, etc.).

Any of a wide variety of cancer therapies can be used in combinationwith the antibody and/or oligosialic acid derivative-based therapiesdescribed herein. Such cancer therapies include surgery (e.g., surgicalremoval of cancerous tissue), radiation therapy, bone marrowtransplantation, chemotherapeutic treatment, biological responsemodifier treatment, and certain combinations of the foregoing.

Radiation therapy includes, but is not limited to, X-rays or gamma raysthat are delivered from either an externally applied source such as abeam, or by implantation of small radioactive sources.

Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous)compounds that reduce proliferation of cancer cells, and encompasscytotoxic agents and cytostatic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents, nitrosoureas,antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, andsteroid hormones.

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (CYTOXAN™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (TAXOL®), docetaxel(TAXOTERE®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-1,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(TAXOL®), TAXOL® derivatives, docetaxel (TAXOTERE®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and ZOLADEX®.Estrogens stimulate proliferation and differentiation, thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include met al complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor;procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); IRESSA® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL, TAXOTERE (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., TAXOTERE™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

In the treatment of some individuals with the compounds of the presentdisclosure, it may be desirable to use a high dose regimen inconjunction with a rescue agent for non-malignant cells. In suchtreatment, any agent capable of rescue of non-malignant cells can beemployed, such as citrovorum factor, folate derivatives, or leucovorin.Such rescue agents are well known to those of ordinary skill in the art.Rescue agents include those which do not interfere with the ability ofthe present inventive compounds to modulate cellular function.

Particular applications in which the methods and compositions find useinclude those described in U.S. Pat. Nos. 2,512,572; 3,892,801;3,989,703; 4,057,548; 4,067,867; 4,079,056; 4,080,325; 4,136,101;4,224,446; 4,306,064; 4,374,987; 4,421,913; 4,767,859; 3,981,983;4,043,759; 4,093,607; 4,279,992; 4,376,767; 4,401,592; 4,489,065;4,622,218; 4,625,014; 4,638,045; 4,671,958; 4,699,784; 4,785,080;4,816,395; 4,886,780; 4,918,165; 4,925,662; 4,939,240; 4,983,586;4,997,913; 5,024,998; 5,028,697; 5,030,719; 5,057,313; 5,059,413;5,082,928; 5,106,950; 5,108,987; 4,106,488; 4,558,690; 4,662,359;4,396,601; 4,497,796; 5,043,270; 5,166,149; 5,292,731; 5,354,753;5,382,582; 5,698,556; 5,728,692; and 5,958,928; the disclosures of whichare herein incorporated by reference.

Production of Anti-Alpha (2→8) or (2→9) Oligosialic Acid DerivativeAntibody Response

Alpha (2→8) or (2→9) oligosialic acid derivatives, including conjugatesthereof, as described herein can be used in eliciting an anti-bacterialantibody response, as well as in eliciting an anti-cancer cell antibodyresponse. In general immunization is accomplished by administration byany suitable route, including administration of the composition orally,nasally, nasopharyngeally, parenterally, enterically, gastrically,topically, transdermally, subcutaneously, intramuscularly, in tablet,solid, powdered, liquid, aerosol form, locally or systemically, with orwithout added excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980).

It is recognized that alpha (2→8) or (2→9) oligosialic acid derivativesand related compounds described herein (e.g., conjugates), whenadministered orally, should be protected from digestion. This istypically accomplished either by complexing the alpha (2→8) or (2→9)oligosialic acid derivative with a composition to render it resistant toacidic and enzymatic hydrolysis or by packaging in an appropriatelyresistant carrier such as a liposome. Means of protecting a compound ofinterest from digestion are well known in the art.

In order to enhance serum half-life, the antigenic preparations that areinjected may also be encapsulated, introduced into the lumen ofliposomes, prepared as a colloid, or other conventional techniques maybe employed which provide an extended serum half-life of the peptides. Avariety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4, 235,871, 4,501,728 and 4,837,028. The preparations may alsobe provided in controlled release or slow-release forms for release andadministration of the antigen preparations as a mixture or in serialfashion.

The compositions are administered to suitable subject, e.g., a subjectthat is at risk from acquiring a Neisserial disease or at risk ofdeveloping a cancer bearing a deNAc SA epitope (e.g., as present in aSEAM 3 and/or DA2-reactive antigen) to prevent or at least partiallyarrest the development of disease and its complications. An amountadequate to accomplish this is defined as a “therapeutically effectivedose.” Amounts effective for therapeutic use will depend on, e.g., theantigen composition, the manner of administration, and a variety ofsubject-specific parameters such as the weight and general state ofhealth of the subject, any or all of which may be modified according tothe judgment of the clinician.

Single or multiple doses of the antigen compositions may be administereddepending on the dosage and frequency required and tolerated by thepatient, and route of administration. In general, immunization isprovided to as to elicit an immune response in the subject, where thecompounds of the present disclosure can provide an advantage thatimmunization does not elicit detectable antibodies that significantlycross-react with polysialic acid in the subject (stated differently,elicits no clinically relevant autoantibody response directed againsthost sialic acid), and can include production of antibodies bactericidalfor N. meningitidis as well as for E. coli K1 and/or production ofantibodies that inhibit cancer cell proliferation.

In particular embodiments, the antigen compositions described herein areadministered serially. First, an immunogenically effective dose of analpha (2→8) or (2→9) oligosialic acid derivative (which may beconjugated to a carrier, and may be with or without excipients) isadministered to a subject. The first dose is generally administered inan amount effective to elicit an immune response (e.g., activation of Band/or T cells). Amounts for the initial immunization generally rangefrom about 0.001 mg to about 1.0 mg per 70 kilogram patient, morecommonly from about 0.001 mg to about 0.2 mg per 70 kilogram patient,usually about 0.005 mg to about 0.015 mg per 70 kilogram patient.Dosages from 0.001 up to about 10 mg per patient per day may be used,particularly when the antigen is administered to a secluded site and notinto the blood stream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages (e.g. 10 to 100 mg or more) arepossible in oral, nasal, or topical administration.

After administration of the first antigen composition of alpha (2→8) or(2→9) oligosialic acid derivative, a therapeutically effective dose of asecond antigen composition (e.g. alpha (2→8) or (2→9) oligosialic acidderivative, optionally conjugated and with or without excipients) isadministered to the subject after the subject has been immunologicallyprimed by exposure to the first dose. The booster may be administereddays, weeks or months after the initial immunization, depending upon thepatient's response and condition.

The presence of a desired immune response may be determined by knownmethods (e.g. by obtaining serum from the individual before and afterthe initial immunization, and demonstrating a change in the individual'simmune status, for example an immunoprecipitation assay, or an ELISA, ora bactericidal assay, or a Western blot, or flow cytometric assay, orthe like) and/or demonstrating that the magnitude of the immune responseto the second injection is higher than that of a control subjectimmunized for the first time with the composition used for the secondinjection (e.g. immunological priming). Immunologic priming and/or theexistence of an immune response to the first antigen composition mayalso be assumed by waiting for a period of time after the firstimmunization that, based on previous experience, is a sufficient timefor an immune response and/or priming to have taken place-e.g. 2, 4, 6,10 or 14 weeks. Boosting dosages of the second antigen composition aretypically from about 0.001 mg to about 1.0 mg of antigen, depending onthe nature of the immunogen and route of immunization.

In certain embodiments, a therapeutically effective dose of a thirdantigen composition prepared from is administered to the subject afterthe individual has been primed and/or mounted an immune response to thesecond antigen composition. The methods disclosed herein alsocontemplate administration of of a fourth, fifth, sixth or greaterbooster immunization, using either a fourth, fifth or sixth antigencomposition.

The subject may be immunologically naive with respect to Neisseriameningitidis or E. coli K1 or a deNAc SA epitope-bearing cancer. Forimmunoprevention, the alpha (2→8) or (2→9) oligosialic acid derivativecan be administered prior the first sign of disease symptoms, or at thefirst sign of possible or actual exposure to infection or disease (e.g.,due to exposure or infection by Neisseria or E. coli K1).

Passive Immunization and Other Antibody-Based Therapies

In addition, anti-alpha (2→8) or (2→9) oligosialic acid derivativeantibodies generated using the methods described herein can be used toprovide for passive immunotherapy, e.g., to treat or prevent N.meningitidis-mediated or E. coli K1-mediated disease in mammaliansubjects. Particularly, the antibodies generated using thede-N-acetylated PS or conjugates thereof according to the presentdisclosure can be provided in a pharmaceutical composition suitable foradministration to a subject, so as to provide for passive protection ofthe subject against N. meningitidis of E. coli K1 disease, or fortreatment of cancer.

More particularly, immunoprotective antibodies generated according tothe methods described herein and that recognize Neisserial PS or E. coliK1 epitopes can be administered to a subject (e.g. a human patient) toinduce passive immunity against a Neisserial disease, either to preventinfection or disease from occurring, or as a therapy to improve theclinical outcome in patients with established disease (e.g. decreasedcomplication rate such as shock, decreased mortality rate, or decreasedmorbidity, such as deafness). Where the antibodies are administered toeffect a cancer therapy, the antibodies can optionally have attached adrug for targeting to the cancer cell to effect tumor killing orclearance, e.g., a toxin (e.g., ricin), radionuclide, and the like).

Diagnostics

Antibodies reactive with a deNAc SA epitope can be used to detect deNAcSA antigens in a biological sample obtained from a subject having orsuspected of having cancerous cells having a cell surface accessibledeNAc SA epitope (e.g., a de-N-acetylated cell surface ganglioside orglycoconjugate) using anti-deNAc SA epitope antibodies inimmunodiagnostic techniques as described in (See U.S. Ser. No.11/645,255 and PCT Application No. US2006/048850; incorporated herein byreference). Such antibodies also find use in detection of deNAc SAantigens in a biological sample obtained from a subject having orsuspected of having bacteria having cell surface accessible deNAc SAepitiopes, e.g., bacteria having polysaccharide containing a deNAc SAepitope, e.g., Neisseria (e.g., Neisseria meningitidis, particularly N.meningitidis Groups B and C), E. coli K1. The present disclosureprovides additional antibodies suitable for this purpose, particularlyin the context of detection of cancer cells given their ability torecognize and bind a deNAc SA epitope on both dividing and non-dividingcells. Such diagnostics can be useful to identify patients amenable tothe therapies disclosed herein, and/or to monitor response to therapy.Further, such antibodies can have or be selected to have antigen-bindingproperties such that the antibodies exhibit little or no detectablebinding to host (e.g., mammalian, especially human) polysialic acid,thereby providing for decreased risk of false positive results.

Briefly, the antigen binding specificity of anti-deNAc SA epitopeantibodies can be exploited in this context, to facilitate detection ofdeNAc SA epitopes on a cancerous or bacterial cell in a sample withlittle or no detectable binding to host-derived PSA, thereby reducingthe incidence of false positive results. Such detection methods can beused in the context of diagnosis, identification of subject suitable toantibody and/or oligosialic acid derivative-based therapy where theantibody specifically binds an deNAc SA epitope and/or a SEAM 3 and/orDA2-reactive antigen, monitoring of therapy (e.g., to follow response totherapy), and the like.

Suitable immunodiagnostic techniques include, but are not necessarilylimited to, both in vitro and in vivo (imaging) methods. Where themethods are in vitro, the biological sample can be any sample in which adeNAc SA antigen may be present, including but not limited to, bloodsamples (including whole blood, serum, etc.), tissues, whole cells(e.g., intact cells), and tissue or cell extracts. Assays can take awide variety of forms, such as competition, direct reaction, or sandwichtype assays. Exemplary assays include Western blots; agglutinationtests; enzyme-labeled and mediated immunoassays, such as ELISAs;biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis;immunoprecipitation, and the like. The reactions generally includedetctable labels such as fluorescent, chemiluminescent, radioactive,enzymatic labels or dye molecules, or other methods for detecting theformation of a complex between antigen in the sample and the antibody orantibodies reacted therewith.

The assays can involve separation of unbound antibody in a liquid phasefrom a solid phase support to which antigen-antibody complexes arebound. Solid supports which can be used include substrates such asnitrocellulose (e.g., in membrane or microtiter well form);polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex(e.g., beads or microtiter plates); polyvinylidine fluoride; diazotizedpaper; nylon membranes; activated beads, magnetically responsive beads,and the like.

Where a solid support is used, the solid support is usually firstreacted with a solid phase component (e.g., an anti-deNAc SA epitopeantibody) under suitable binding conditions such that the component issufficiently immobilized to the support. Sometimes, immobilization tothe support can be enhanced by first coupling the antibody to a proteinwith better binding properties, or that provides for immobilization ofthe antibody on the support with out significant loss of antibodybinding activity or specificity. Suitable coupling proteins include, butare not limited to, macromolecules such as serum albumins includingbovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulinmolecules, thyroglobulin, ovalbumin, and other proteins well known tothose skilled in the art. Other molecules that can be used to bindantibodies the support include polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andthe like, with the proviso that the molecule used to immobilize theantibody does not adversely impact the ability of the antibody tospecifically bind antigen. Such molecules and methods of coupling thesemolecules to the antigens, are well known to those of ordinary skill inthe art. See, e.g., Brinkley, M. A. Bioconjugate Chem. (1992) 3:2-13;Hashida et al., J. Appl. Biochem. (1984) 6:56-63; and Anjaneyulu andStaros, International J. of Peptide and Protein Res. (1987) 30:117-124.

After reacting the solid support with the solid phase component, anynon-immobilized solid-phase components are removed from the support bywashing, and the support-bound component is then contacted with abiological sample suspected of containing deNAc SA epitopes undersuitable binding conditions. After washing to remove any non-boundligand, a secondary binder moiety is added under suitable bindingconditions, wherein the secondary binder is capable of associatingselectively with the bound ligand. The presence or absence of thesecondary binder can then be detected using techniques well known in theart.

An ELISA method can be used, wherein the wells of a microtiter plate arecoated with anti-deNAc SA epitope antibody according to the presentdisclosure. A biological sample containing or suspected of containing adeNAc SA antigen (e.g., a tumor antigen having a deNAc SA epitope, suchas a de-N-acetylated ganglioside), is then added to the coated wells.After a period of incubation sufficient to allow antibody binding, theplate(s) can be washed to remove unbound moieties and a detectablylabeled secondary binding molecule added. The secondary binding moleculeis allowed to react with any captured antigen, the plate washed and thepresence or absence of the secondary binding molecule detected usingmethods well known in the art.

Where desired, the presence or absence of bound deNAc SA antigen from abiological sample can be readily detected using a secondary bindercomprising an antibody directed against the antibody ligands. Forexample, a number of anti-bovine immunoglobulin (Ig) molecules are knownin the art which can be readily conjugated to a detectable enzyme label,such as horseradish peroxidase, alkaline phosphatase or urease, usingmethods known to those of skill in the art. An appropriate enzymesubstrate is then used to generate a detectable signal. In other relatedembodiments, competitive-type ELISA techniques can be practiced usingmethods known to those skilled in the art.

Assays can also be conducted in solution, such that the antibodies anddeNAc SA antigen form complexes under precipitating conditions. Forexample, the antibody can be attached to a solid phase particle (e.g.,an agarose bead or the like) using coupling techniques known in the art,such as by direct chemical or indirect coupling. The antibody-coatedparticle is then contacted under suitable binding conditions with abiological sample suspected of containing deNAc SA antigen to providefor formation of particle-antibody-deNAc SA antigen complex aggregateswhich can be precipitated and separated from the sample using washingand/or centrifugation. The reaction mixture can be analyzed to determinethe presence or absence of antibody-antigen complexes using any of anumber of standard methods, such as those immunodiagnostic methodsdescribed above.

The test sample used in the diagnostics assays can be any sample inwhich a deNAc SA antigen may be present, including but not limited to,blood samples (including whole blood, serum, etc.), tissues, whole cells(e.g., intact cells), and tissue or cell extracts containing cells(e.g., tissue, isolated cells, etc.), a cell lysate (i.e., a samplecontaining non-intact cells), where each type of sample can containelements of both types (e.g., a sample of cells can contain celllysates, and vice versa). In some embodiments, particularly as inembodiments involving detection of cancer cells, it may be desirable toconduct the assay using a sample from the subject to be diagnosed thatcontains intact, living cells. DeNAc SA antigen detection can then beassessed on an extracellular surface of the cells, and can further beassessed during cell division.

Diagnostic assays can also be conducted in situ. For example, anti-deNAcSA epitope antibodies can be detectably labeled, administered to asubject suspected of having a cancer characterized by cell surfaceexpression of a deNAc SA epitope, and bound detectably labeled antibodydetected using imaging methods available in the art.

The diagnostic assays described herein can be used to determine whethera subject has a cancer that is more or less amenable to therapy usingantibody and/or oligosialic acid derivative-based therapy, as well asmonitor the progress of treatment in a subject. It also may be used toassess the course of other combination therapies (e.g., deNAc SA antigenvaccine and/or anti-deNAc SA antigen antibody therapy as described in(U.S. Ser. No. 11/645,255 and PCT Application No. US2006/048850;incorporated herein by reference). Thus, the diagnostic assays caninform selection of therapy and treatment regimen by a clinician.

Where the methods are in vitro, the biological sample can be any samplein which a SEAM 3 and/or DA2-reactive antigen may be present, includingbut not limited to, blood samples (including whole blood, serum, etc.),tissues, whole cells (e.g., intact cells, i.e., cells that have not beensubjected to permeabilization), or cell lysates (e.g., as obtained fromtreatment of a tissue sample). For example, the assay can involvedetection of a SEAM 3 and/or DA2-reactive antigen on cells in ahistological tissue sample. For example, the tissue sample may be fixed(e.g., by formalin treatment) and may be provided embedded in a support(e.g., in paraffin) or frozen unfixed tissue.

The SEAM 3 and/or DA2-reactive antigen can be detected by detection ofspecific binding of an antibody, usually a monoclonal antibody (mAb),that has the antigen-binding specificity of SEAM 3 and/or DA2. In thisembodiment, the SEAM 3 and/or DA2-reactive antigen may be present on thecell surface at any stage of the cell cycle, including during celldivision. Of note is that in some instances, cancers that present a SEAM3 and/or DA2-reactive antigen during cell division may present a loweror no detectable level of SEAM 3 and/or DA2-reactive antigen when thecell is quiescent (i.e., not undergoing cell division). However, asillustrated in the examples below, SEAM 3 and/or DA2-reactive antigencan be detected in non-dividing cells by detecting SEAM 3 and/orDA2-reactive antigen in a permeabilized test cell. A test cancer cellthat exhibits a pattern of staining with a SEAM 3 and/or DA2 antibody(or an antibody having the antigen binding specificity of SEAM 3 and/orDA2) that is distinct from a pattern of antibody staining in a normalcell is identified as a cancerous cell that exhibits a SEAM 3 and/orDA2-reactive antigen. Such cancers are thus amenable to therapy with anantibody that specifically binds the SEAM 3 and/or DA2-reactive antigen(e.g., the mAb SEAM 3 and/or the mAb DA2).

The above-described assay reagents, including the antibodies generatedby immunization with a deNAc SA antigen according to the methodsdescribed in U.S. Ser. No. 11/645,255 and PCT Application No.US2006/048850, can be provided in kits, with suitable instructions andother necessary reagents, in order to conduct immunoassays as describedabove. The kit can also contain, depending on the particular immunoassayused, suitable labels and other packaged reagents and materials (i.e.wash buffers and the like). Standard immunoassays, such as thosedescribed above, can be conducted using these kits.

Kits & Systems

Also provided are kits and systems that find use in practicing themethods, as described above. For example, kits and systems forpracticing the methods may include one or more pharmaceuticalformulations that include antibody and/or oligosialic acid derivative.As such, in certain embodiments the kits may include a singlepharmaceutical composition present as one or more unit dosages. In yetother embodiments, the kits may include two or more separatepharmaceutical compositions.

Thus the kits can include one or more of, depending upon the intendeduse of the kit, the compositions described herein, such as: anoligosialic acid derivative and/or antibody specific thereto, cellssuitable related for assays or screening, an anti-deNAc SA epitopeantibody, and the like. Other optional components of the kit include:buffers, etc., for administering an oligosialic acid derivative and/orantibody specific thereto, and/or for performing a diagnostic assay. Thevarious components of the kit may be present in separate containers orcertain compatible components may be pre-combined into a singlecontainer, as desired.

In addition to the above components, the kits may further includeinstructions for practicing the methods. These instructions may bepresent in the kits in a variety of forms, one or more of which may bepresent in or on the kit. One form in which these instructions may bepresent is as printed information on a suitable medium or substrate,e.g., a piece or pieces of paper on which the information is printed, inor on the packaging of the kit, in a package insert, etc. Yet anothermeans would be a computer readable medium, e.g., diskette, CD, etc., onwhich the information has been recorded. Yet another means that may bepresent is a website address which may be used via the internet toaccess the information at a removed site. Any convenient means may bepresent in the kits.

In a specific embodiment, a kit is provided for use in treating a hostsuffering from a cellular proliferative disease condition. This kitincludes a pharmaceutical composition comprising an oligosialic acidderivative and/or antibody specific thereto, and instructions for theeffective use of the pharmaceutical composition in a method of treatinga host suffering from a cancerous condition by inhibiting the growth ofa cancer cell in a subject, or by providing for an anti-deNAc SA immuneresponse, e.g., to elicit antibodies that bind a cancer cell bearing adeNAc SA epitope. Such instructions may include not only the appropriatehandling properties, dosing regiment and method of administration, andthe like, but can further include instructions to optionally screen thesubject for a de-N-acetylated sialic acid (deNAc SA) epitope. Thisaspect can assist the practitioner of the kit in gauging the potentialresponsiveness of the subject to treatment with an oligosialic acidderivative and/or antibody specific thereto, including timing andduration of treatment relative to the type and growth stage of thecancer. Thus in another embodiment, the kit may further include anantibody or other reagent for detecting a de-N-acetylated sialic acid(deNAc SA) epitope on an extracellularly accessible surface of a cancercell, such as SEAM 3 and/or DA2. In another embodiment, the kit includesone or more alpha (2→8) or (2→9) oligosialic acid derivatives thatcomprise a conjugate with a detectable label, such as a fluorophore.

In another specific embodiment, a kit is provided for use in immunizinga host at risk of, or having, a disease or disease symptom of infectionby a bacteria bearing a deNAc SA epitope, e.g., a deNAc SA epitope on abacterial polysaccharide capsule (e.g., Neisseria (e.g., N.meningitidis, especially Groups B and C N. meningitidis), E. coli K1).This kit includes a pharmaceutical composition comprising an oligosialicacid derivative and/or antibody specific thereto, and instructions forthe effective use in immunization or treatment of a host having, or atrisk of, bacterial infection. Such instructions may include not only theappropriate handling properties, dosing regiment and method ofadministration, and the like, but can further include instructions tooptionally screen the subject for a de-N-acetylated sialic acid (deNAcSA) epitope. This aspect assists the practitioner of the kit in gaugingthe potential responsiveness of the subject to immunization with anoligosialic acid derivative and/or antibody specific thereto. Thus inanother embodiment, the kit may further include an antibody or otherreagent for detecting a de-N-acetylated sialic acid (deNAc SA) epitopeon an extracellularly accessible surface of a cancer cell, such as SEAM3 and/or DA2.

The term “system” as employed herein refers to a collection of anoligosialic acid derivative and/or antibody specific thereto and one ormore second therapeutic agents, present in single or disparatecompositions that are brought together for the purpose of practicing themethods. For example, separately obtained oligosialic acid derivativeand/or antibody specific thereto and chemotherapy dosage forms broughttogether and coadministered to a subject are a system according to thepresent disclosure.

The following examples further illustrate the present invention andshould not be construed as in any way limiting its scope.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Example 1 Preparation of Alpha (2→8) N-Acetyl Neuraminic AcidOligosaccharides (OS)

Colominic acid (100 mg, Sigma-Aldrich, Saint Louis, Mo.), which is PSAisolated from the capsule of Escherichia coli K1 bacteria, was dissolvedin 5 ml of 20 mM HCl and heated to 50° C. for 2 hrs. After cooling toambient temperature, the pH was increased to 8-9 with 2M NaOH. Thesolution was dialyzed (1 kDa cutoff tubing, Spectrapor obtained fromThermo-Fisher Scientific, Waltham, Mass.) in water and lyophilized.

Example 2 Sodium Borohydride Treatment of Oligosaccharides

The lyophilized OS (100 mg) from Example 1 were combined with 10 mg ofsodium borohydride (Sigma-Aldrich) in 5 ml of water and left at ambienttemperature overnight. Over the course of several hours, the pH of thesolution rises from approximately 8.5 to approximately 10. The reactionmixture was dialyzed in water and lyophilized as described above inExample 1. The resulting OS antigen was determined to contain about 33%neuraminic acid residues, and contain a mixture of chains having adegree of polymerization of about 2-20.

Example 3 Analysis of Sodium Borohydride-Treated Oligosaccharides

Sodium borohydride-treated OS of Example 2 were separated by ionexchange chromatography on an Äkta™ FPLC fitted with a 5 ml HiTrap Q FF™anion exchange column (GE Healthcare Bio-Sciences Corp., Piscataway,N.J.). 20 mg of OS were dissolved in 0.5 ml of 20 mM Bis-Tris buffer(Sigma-Aldrich), pH 8 and injected onto the column. OS were eluted fromthe column with a OM to 0.5M gradient of sodium sulfate in 20 mMBis-Tris buffer. The amount of sialic acid and de-N-acetyl sialic acidin each 1 ml fraction was determined by resorcinol assay described inExample 6, below. Also, the ability of each fraction to inhibit bindingof SEAM 3 to N-propionyl NmB polysaccharide dodecylamine was determinedby inhibition ELISA as described in Example 5, below.

The results are summarized in the graph shown in FIG. 1. Essentially allof the OS retained by the column contained both sialic acid andde-N-acetyl sialic acid. The ratio ranged from roughly 3:1 for the shortoligosaccharides eluting at low salt to 10:1 or more for the longerpolysaccharides eluting with higher salt concentrations. Also, allfractions containing a mixture of sialic acid and de-N-acetyl sialicacid inhibited SEAM 3 binding.

After dialysis in water and lyophilization each fraction contained 1 mgor less of OS. Treatment of selected fractions with excess amounts ofthe exoneuraminidase SIALIDASE A (Prozyme, San Leandro, Calif.) did notdecrease the amount of OS or affect the ability of the OS to inhibitSEAM 3 binding. Since exoneuraminidases are unable to degrade PSA thatterminate at the non-reducing end with a de-N-acetyl residue (i.e.neuraminic acid), the results suggest that de-N-acetylation resultingfrom sodium borohydride treatment is occurring, at least in part if notwholly at the non-reducing end of the OS.

It is also possible that borohydride, boranes, or borates produced byreaction of borohydride with the OS results in the formation of boron-OScomplexes. However, several samples of OS derivatives that are goodinhibitors of SEAM 3 binding were tested for the presence of boron byazomethine (Sigma-Aldrich) colormetric assay (Zenki et al, Fresenius' JAnal Chem, 1989, 334:238) and by inductively coupled plasma massspectroscopy (performed by Galbraith Laboratories, Inc., Knoxville,Tenn. An amount corresponding to mole fraction of less than 1% could bedetected.

The OS fractions were further analyzed by high performance anionexchange chromatography with pulsed ampermetric detection (HPAC-PAD) todetermine the length of OS in each fraction. The results for selectedfractions are shown in FIG. 2. The fraction having shortest OS that werestill able to inhibit SEAM 3 binding were in fraction 29 which containeda mixture of degree of polymerization (Dp) of 2 to 6 but mostly 4 to 6(FIG. 2).

Example 4 Preparation of Dodecylamine Derivatives of N-Propionyl NMB PS

Twenty (20) mg of N-propionyl NmB polysaccharide (N-Pr NmB PS) oxidizedwith periodate prepared as described by Granoff et al (Granoff et al.,J. Immunol, 1998, 160: 5028) was combined in water (5 ml) with 5 μl ofdodecylamine (Thermo-Fisher). The pH was adjusted to 8 with 2M HCl andthe mixture stirred for 3 hrs. Sodium cyanoborohydride (5 mg,Sigma-Aldrich) was added and the mixture was stirred at ambienttemperature for 24 hours then dialyzed in water for 3 to 5 days toremove excess dodecylamine. The dodecylamine antigen (˜1 mg/ml in PBSbuffer) was stored at 4° C.

Example 5 Seam 3 Inhibitor Assay

ELISA plates for testing inhibitors of SEAM 3 binding were prepared bydiluting the dodecylamine N-propionyl NmB derivative of Example 4 above1:200 in PBS buffer and adding 100 μl per well of a 96 well microtiterplate (Immulon II HB, Dynatech, Chantilly, Va.). The plates were storedovernight at 4° C. before use in assays. The plates were washed with PBSbuffer (5×) and blocked with PBS buffer containing 1% (weight/volume) ofbovine serum albumin (Sigma; blocking buffer) for 1 hour at ambienttemperature. The inhibitors were diluted in blocking buffer on the platethen SEAM 3 was added in blocking buffer (100 μl total per well). Afterincubating the plate overnight at 4° C., the plates were washed with PBSbuffer (5×) and rabbit anti-mouse-alkaline phosphatase conjugateantibody (Zymed, South San Francisco, Calif.) diluted in blocking bufferwas added. After incubating an additional hour, the plates were washed(5×) with PBS buffer and the bound antibody was detected by addingp-nitrophenyl phosphate substrate (Sigma-Aldrich) in 50 mM sodiumcarbonate buffer, pH 9, containing 1 mM MgCl₂. The absorbance at 405 nmafter 60 minutes incubation at ambient temperature was measured using aBioRad Model 550 microtiter plate reader (Richmond, Calif.).

Example 6 Determination of Sialic Acid and Neuraminic Acid in PSA

The concentration of sialic acid and de-N-acetyl sialic acid in PSAderivative stock solutions or column fractions was determined by theSvennerholm resorcinol reaction (Svennerholm, L. (1957) Biochim.Biophys. Acta 24:604) modified as follows. Resorcinol working reagentwas prepared by combining 9.75 ml of water, 0.25 ml of 0.1 M CuSO4.5H2O,10 ml of 20 milligram per ml solution of resorcinol in water, and 80 mlof concentrated HCl. The resorcinol working reagent (300 μl) wascombined with the sialic acid or de-N-acetyl sialic acid sample solution(up to 50 micrograms of sialic acid) or standard stock solution in water(300 μl) in a polypropylene deep well (2 ml) microtiter plate. The platewas sealed with a plate cover and heated in a boiling water bath for 30minutes. After cooling to ambient temperature, isoamyl alcohol (600 μl)was added and mixed using a pipette. The phases were allowed to separateand the upper isoamyl alcohol layer was removed to a clean microtiterplate. 250 μl of the isoamyl alcohol extract and the lower aqueoussolution were transferred separately to a polystyrene microtiter plateand the absorbance at 495 nm and 580 nm was measured.

The amount of N-acetyl sialic acid was determined from the absorbance ofthe isoamyl alcohol fraction at 580 nm and the amount of de-N-acetylsialic acid was determined from the absorbance of the aqueous fractionat 495 nm in comparison to a standard curve for each. The amount ofde-N-acetyl sialic acid was corrected for the amount of de-N-acetylationthat occurs during the acid hydrolysis step of the assay by measuringthe amount of de-N-acetylation that occurs in the sialic acid standard.

Example 7 Determining the Minimal Length of Oligosaccharide forReactivity with SEAM 3

To prepare a vaccine that is intended to elicit antibodies and havespecificities similar to that of SEAM 3 it is necessary to determine theminimal alpha (2→8) neuraminic acid OS length that is reactive with SEAM3. Longer PSA has the potential to elicit antibodies that are reactivewith other human PSA antigens. To determine the minimal length OSrecognized by SEAM 3, N-acetyl neuraminic acid monomer, and alpha (2→8)linked dimer, trimer, and tetramer (10 mg each, EY ScientificLaboratories, San Mateo, Calif.) were combined with 1 mg of sodiumborohydride in water as described above. After dialysis andlyophilization, each oligomer was tested by ELISA for the ability toinhibit binding of SEAM 3 to N-Pr NmB PS-dodecylamine. The results aresummarized in Table 1. None of the untreated control OS (i.e., OS thathad not been treated with sodium borohydride) were able to inhibit SEAM3 binding. As shown in Table 1, a borohydride-treated oligosaccharide asshort as a tetramer exhibits all of the activity of the much longernominal polysaccharide antigen N-Pr NmB PS.

TABLE 1 Specificity of SEAM 3 determined by inhibition ELISA InhibitorDp* [IC₅₀] (μg/ml)+ N-Propionyl PSA >20 0.08 Re-N-Acetyl PSA >20 1.6Tetramer 3 0.02 Trimer 2 1.9 Dimer 1 51 NmC PS# >20 >200 *Excluding thereducing end residue, which is reduced and the non-reducing end residuethat may be de-N-acetylated and/or form a complex with boron.+Concentrations of sialic acid in the stock solutions used in theinhibition experiments were determined by resorcinol assay. #NeuNmC PS,NmW PS and NeuNmW PS were also tested with similar results to NmC PS(NmC PS, and NmW PS are N. meningitidis group C and W, respectively,capsular polysaccharides)

Example 8 Preparation of a OS-Tetanus Toxoid Conjugate Vaccine

Sodium borohydride-treated OS prepared as described above (20 mg) wasoxidized with sodium periodate (6 μmol or 1 equivalent for every 10residues) for 1 hr in 2 ml of 0.1M sodium acetate buffer, pH 6.5.Ethylene glycol (100 μl of a 10% (volume/volume) solution in water) wasadded to destroy any remaining periodate and the solution was dialyzedin water. OS (10 mg) was combined with 5 mg of tetanus toxoid (BioVerisCorp., Gaithersburg, Md.) in 5 ml of PBS buffer. The solution wasstirred overnight at ambient temperature in a brown glass Reacti-Vial(Pierce Chemical Company, Rockford, Ill.). The following day, 5 mg ofsodium cyanoborohydride was added and stirring of the mixture wascontinued for 2 days. The reaction mixture was dialyzed (10-14 kDacutoff membrane) in PBS buffer. The vaccine preparation was sterilefiltered (0.22 μ), aliquoted and stored at −80° C. until used. Thevaccine solution contained 2.4 mg/ml sialic acid and 1.2 mg/mlde-N-acetyl sialic acid determined by resorcinol assay and 1.5 mg/mlprotein as determined by BCA assay (Bio-Rad). To demonstrate that the OSantigen was covalently linked to tetanus toxoid, a portion of thevaccine was separated on a 4%-15% sodium dodecyl sulfate-polyacrylamidegel (Bio-Rad) and tested for reactivity with SEAM 3 by Western blot.Bound SEAM 3 was detected using a rabbit anti-mouse IgG polyclonalantibody conjugated to horse radish peroxidase (Zymed) and WesternLighting® chemiluminescence reagents (PerkinElmer Life and AnalyticalSciences, Waltham, Mass.). As shown in FIG. 3, SEAM 3 binds to the highmolecular weight tetanus toxoid derivative running at the top of thegel. The tetanus toxoid derivative is referred to herein as “OS-tetanustoxoid” conjugate.

Example 9 Evaluating the Immunogenicity in CD1 Mice

OS-tetanus toxoid conjugates prepared above were used to evaluateimmunogenicity in CD1 mice as follows. Groups of 10 female CD1 mice (6-8weeks old, Charles River Laboratories, Wilmington, Mass.) were immunizedwith either 2 μg or 10μg of total (i.e. N-acetyl plus de-N-acetyl)sialic acid OS-tetanus toxoid conjugate vaccine in 50% 0.9% saline/50%Freund's complete adjuvant (Pierce) emulsion by ip injection. Ten daysafter the first dose, blood samples were obtained by lancet of thesubmandibular vein and tested by ELISA using OS conjugated to bovineserum albumin (BSA, Pierce) (referred to as “DeNAc-BSA”) prepared asdescribed above for the OS-tetanus toxoid conjugate. Four weeks afterthe first dose, a second dose was given with Incomplete Freund'sadjuvant (Pierce). Again, 10 days after the second dose, blood sampleswere obtained from the mice for testing.

FIG. 4 shows the mean titers for each group determined by ELISA using OSconjugated to bovine serum albumin (BSA, Imject, Pierce Chemical Co.,Rockford, Ill.) as described in Example 14). The immune response to 2 μgand 10 μg doses were similar and increase approximately 100-fold betweenthe primary and booster doses. The result shows that the vaccine ishighly immunogenic and elicits an antigen-specific antibody response.

Example 10 Evaluating Functional Activity of Post-2^(nd) Dost Antiserawith Neisseria meningitidis Group B Bacteria

The ability of antibodies elicited by immunization with OS-tetanustoxoid conjugate vaccine to activate deposition of human complementcomponents on NmB bacteria was determined as described by Welsch et al(Welsch et al, J Infec Dis, 2003, 188:1730). Briefly, the NmB strain NMBwas grown to an OD620 nm of 0.6 in Muller-Hinton media containing 0.3%glucose. The cells were pelleted and washed with Dulbecco's PBS(Invitrogen, Carlsbad, Calif.) containing 1% (weight/volume) BSA(Sigma-Aldrich) (D-BSA) and resuspended in half the volume D-BSA of theoriginal growth media. The bacteria (30 μl) were combined with thepooled antisera from mice immunized with either 2 μg or 10 μg wasdiluted to 1:20 or 1:200 in D-BSA. Human complement from a donor testedfor the absence of antibodies to NMB was added to a concentration of 5%(volume/volume) in a total volume of 200 μl. The reaction was allowed toproceed for 30 minutes at ambient temperature with occasional agitation.The cells were pelleted, washed with 200 μl of D-BSA and FITC-conjugatedsheep anti-human C3c antibody (BioDesign International, Saco, Me.) wasadded in D-BSA. After 30 minutes incubation on ice with occasionalagitation, the cells were pelleted, resuspended in sterile filtered PBSbuffer containing 0.5% (weight/volume) formaldehyde and analyzed by flowcytometery (BD FACSCalibur System, BD Biosciences, San Jose, Calf.). Theresults are shown in FIG. 5.

Deposition of complement components on the cell surface increases thefluorescence of the cells and is indicated by the shift in fluorescencepeak to the right of the graph. The antibody activation is indicated bythe lack of fluorescence of cells alone with active complement orantisera with heat-inactivated complement. The bacteria from flowcytometry were analyzed further by microscopy using a Zeiss Axioplan(Carl Zeiss, Inc.) fluorescence microscope. FIG. 6 shows a micrograph(200×) of bacterial cells after incubation with complement and pooledantisera diluted 1:200 from CD1 mice immunized with a 10 μg booster doseof the OS-tetanus toxoid conjugate vaccine stained with theFITC-conjugated sheep anti-human C3c antibody. In this cluster of cells,numerous highly fluorescent diplococci can be seen. The high level offluorescence indicates the presence of complement factors that mediatebacteriolysis and opsonophagocytosis bound to the surface of the cells.In contrast, no fluorescent cells were observed in the negative controls(cells only with complement or antisera with heat inactivatedcomplement). Activation of complement factor deposition on the cellsurface of NmB bacteria is correlated with protection against diseasecaused by NmB (Welsch et al, J Infec Dis, 2003, 188:1730).

An alternative approach to evaluating the ability of antibodies elicitedby a vaccine to protect against disease caused by NmB is to determinewhether the antisera can lyse or inhibit the growth of NmB ex vivo inhuman blood. In this experiment, antisera (pooled antisera from CD1 miceimmunized with 10 pg of OS-tetanus toxoid conjugate vaccine) and thetest bacteria (approximately 1000 CFU of NmB strain NZ98/254 freshlygrown in Muller-Hinton media as described above) are combined in freshlyobtained human blood from a donor who lacks antibodies to the teststrain in sterile glass vials. The blood is drawn from the donor withthe thrombin inhibitor hirudin (50 mg/ml, 1 μl per ml of blood drawn,Refludan®, Berlex Laboratories, Montville, N.J.) in the syringe needle.Aliquots and diluted aliquots of the mixture are plated onto chocolateagar plates (Remel, Lenexa, Kans.) to determine the CFU/ml at the startof the experiment and at 1 hr and 2 hr intervals. The results of testingthe antisera in the ex vivo human blood model of meningococcalbacteremia are shown graphically in FIG. 7.

In the absence of antibody or in the presence of 50 μg/ml of a negativecontol mAb anti-porin P1.2, an initial inoculation of about 1500 CFUincreases to ˜7000 CFU at 1 hr and ˜60,000 CFU after 2 hrs. However, inthe presence of 2 μg/ml of the positive control mAb, anti-porin P1.4,all of the bacteria are killed. Similarly, both 1:25 and 1:200 dilutionsof the test vaccine antisera result in a decrease in the number ofviable bacteria compared to the controls of 10-fold. While the CFU/ml attime 0 and at 1 hr are approximately the same as the negative controlreactions, after 2 hrs no further growth of the bacteria is observed(˜7000 CFU/ml). The result shows that the antibody activates protectivemechanisms present in human blood (complement mediated bacteriolysisand/or opsonophagocytosis) that decreases the viability of the bacteriain human blood. Thus, antibodies elicited by the vaccine describedherein have the potential to protect against disease caused by NmB.

Example 11 Binding of Vaccine Elicited Antibodies to PSA DerivativesExpressed by the Jurkat T-Cell Leukemia Cell Line

SEAM 3 binds to neuraminic acid-containing PSA antigens expressed by theT-cell leukemia cell line Jurkat (U.S. Ser. No. 11/645,255 and PCTApplication No. US2006/048850; incorporated herein by reference). Tomeasure binding of antibodies elicited by OS-conjugate vaccine, Jurkatcells were spun at 1000×g for 5 minutes and fixed with ice-cold 1% (v/v)formaldehyde. After 20 minutes the cells were pelleted by centrifugation(as above) and incubated in a blocking solution of 3% (v/v) goat serumfor 1 hour. After blocking, the pooled antisera from CD1 mice immunizedwith the 2 doses of 2 μg of total sialic acid OS-tetanus toxoidconjugate vaccine was added and incubated overnight at 4° C. The cellswere washed twice by pelleting and resuspension in ice-cold PBS.Secondary antibody (FITC-conjugated goat anti-mouse IgG (Fab)₂, JacksonImmunoresearch, West Grove, Pa.) was incubated with the cells for atleast 1 hour at 4° C. in the dark. After another series of spins andwashes (3 times) binding was analyzed by a Guava EastCyte flow cytometer(Guava Technologies, Hayward, Calif.). Control samples were treated withan isotype matched irrelevant antibody (Southern Biotech, Birmingham,Ala.), which was used to create baseline fluorescence. The results ofthe Jurkat cell binding experiment are shown in FIG. 8.

Antibody binding to the cells is indicated by the shift to greaterfluorescence (shift to the right of the histogram). Less than 5% ofcells are positive for binding with the negative control irrelevantisotype matched IgG2b mAb. The positive control mAb SEAM 3 bindingresults in a small shift to higher fluorescence (mean fluorescence 210AU) and 12% of the cells are positive for binding. Only a fraction ofthe cells are positive as the antigen is expressed on the surface ofcells mainly during cell division. In contrast, binding of antibodiesfrom the post-boost pooled antisera from the 2 μg dose of OS-tetanustoxoid conjugate diluted 1:20 results in a large increase influorescence (450 AU) and 35% of the cells are positive. Thus, theOS-tetanus toxoid conjugate vaccine elicits antibodies that are reactivewith neuraminic acid-containing antigens expressed by Jurkat T-cellleukemia cells.

Example 12 Preparation of DeNac, NPrSia and TcAc Vaccine Antigens

De-N-acetylpoly α(2→8) neuraminic acid (DeNAc) PSA. Colominic acid (100mg, Sigma-Aldrich) and sodium borohydride (10 mg) were suspended inwater (8.8 ml). After adding NaOH (1.8 ml of 50% solution;Thermo-Fisher) to a final NaOH concentration of 2M, the solution washeated to between 90° C. and 100° C. for 2 hrs. After cooling thesolution to ambient temperature, 2M HCl was added to adjust the pH to 8.Precipitates were removed by centrifugation, the supernatant solutionwas dialyzed two times in 4L of water (1 kDa Spectrum Spectra/Por* 7dialysis membrane; Thermo-Fisher) and lyophilized. The resulting DeNAcantigen was determined to contain about 98% neuraminic acid residues(i.e., de-N-acetyl neuraminic acid or “Neu”), and contains a mixture ofchains having a degree of polymerization of about 2-20.

N-Trichloroacetyl (TcAc) PSA. DeNAc PSA (50 mg) was suspended in water(5 ml) and the pH adjusted to 8-9 with 2M NaOH. Trichloroacetyl chloride(Sigma-Aldrich) was added to the stirred solution in 5 0.1 ml aliquotsover a period of 1 hr. The pH was maintained between 8 and 9 by adding 2M NaOH. The reaction mixture was dialyzed in water and lyophilized asdescribed above. The resulting TcAc antigen was determined to containabout 63% neuraminic acid residues, and contain a mixture of chainshaving a degree of polymerization of about 2-20.

N-propionyl (NPr) PSA and sialic acid-treated N-propionyl (NPrSia) PSANPr PSA was prepared as described for TcAc PSA except that propionicanhydride (Sigma-Aldrich) was used in place of the acid chloride. Theresulting NPr antigen was determined to contain about 21% neuraminicacid residues, and contain a mixture of chains having a degree ofpolymerization of about 30.

Exoneuraminidases are unable to degrade or degrade much slower PSA thatcontains neuraminic acid at the non-reducing end (T. Bhandari and G.Moe, unpublished). Therefore, a portion (20 mg) of NPr PSA was furthertreated with the exoneuraminidase SIALIDASE A (Prozyme) to increase thefraction of molecules that terminate at the non-reducing end inneuraminic acid. The polysaccharide was incubated with SIALIDASE A (10μl of 1 U/ml stock, Prozyme) in the 50 mM sodium phosphate buffer, pH 7at 37° C. for two days. The reaction mixture was dialyzed in water andlyophilized as described above. The resulting NPrSia antigen wasdetermined to contain about 7% neuraminic acid residues, and contain amixture of chains having a degree of polymerization of about 2-20.

Example 13 Preparation of Tetanus Toxoid Conjugate (PS-TT) Vaccines

DeNAc, NPr, NPrSia, and TcAc antigens were oxidized with periodate andconjugated to tetanus toxoid (TT) as in Example 8 The PS-TT vaccinepreparations (DeNAc-TT, NPr-TT, NPrSia-TT, TcAc-TT and OS-TT) weresterile filtered (0.22 μ), aliquoted and stored at −80° C. until used.The composition of the vaccine solutions are summarized in Table 2.NeuNAc (N-acetyl neuraminic acid) and Neu (de-N-acetyl neuraminic acid)were determined by resorcinol assay as described in Example 6. Theprotein concentration was determined by BCA assay (Bio-Rad).

To demonstrate that the antigens were covalently linked to TT and tocompare the reactivity of the conjugate vaccines with non-autoreactivemAbs SEAM 2 and 3 and autoreactive mAb SEAM 18 (Granoff et al., J.Immunol, 1998, 160: 5028), a portion of the vaccines were separated on a4%-15% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad) and testedfor reactivity with the mAbs by Western blot. Bound mAb was detectedusing a rabbit anti-mouse IgG polyclonal antibody conjugated to horseradish peroxidase (Zymed) and Western Lighting® chemiluminescencereagents (PerkinElmer Life and Analytical Sciences, Waltham, Mass.). Asshown in FIG. 9, SEAM 2 binds to the TcAc-TT and NPr-TT conjugatevaccines (high molecular weight derivative running at the top of thegel), SEAM 3 binds to the NPr-TT and NPrSia-TT, and OS-TT conjugatevaccines, and SEAM 18 binds to the NPr-TT conjugate vaccine. None of themAbs bind to the DeNAc-TT conjugate vaccine.

TABLE 2 NeuNAc, Neu, and protein composition of PS-TT vaccines. NeuNAcNeu Percent Total Sialic Acid Protein Vaccine (mg/ml) (mg/ml) Neu(mg/ml) (mg/ml) NPr-TT 3.3 0.9 21 4.2 3.1 NPrSia-TT 4.2 0.3 7 4.5 3.1OS-TT 2.4 1.2 33 3.6 2.4 TcAc-TT 2.2 3.7 63 5.9 3.6 DeNAc-TT 0.2 7.8 988 5.1

Example 14 Evaluating the Immunogenicity of PS-TT Vaccines in CD1 Mice

The immunogenicity of the PS-TT conjugates prepared in Example 13 wasevaluated in CD1 mice as follows. Groups of 10 female CD1 mice (6-8 wkold, Charles River Laboratories, Wilmington, Mass.) were immunized witheither 2 μg or 10 μg of total (i.e. N-acyl plus de-N-acetyl) sialicacid-TT conjugate vaccine in 50% 0.9% saline/50% Freund's completeadjuvant (Pierce) emulsion by ip injection. Blood samples were obtainedby lancet of the submandibular vein 10 days after each injection andtested by ELISA.

Booster doses were given at post 28 days with incomplete Freund'sadjuvant (Pierce) and titers of antisera obtained 10 days postimmunization were evaluated. After 56 days post primary immuniation, thegroups were split in half. Five mice from each group were givenunconjugated PS and the other 5 conjugated PS, both without adjuvant.Since the immune response of the mice that had received the unconjugatedPS was very weak, they were given a dose 3 dose of conjugate withoutadjuvant 112 days post primary immunization. Antisera from this fourthdose are designated 3-PS throughout, where 3 indicates the 3dimmunization.

FIG. 10 shows the mean titers for each group determined by ELISA. Theantibody titer elicited by the PS-TT vaccines was measured by ELISAusing PS conjugated to BSA (PS-BSA) prepared as described above for thePS-tetanus toxoid conjugates (Example 8). Initially, antiserum from eachmouse was tested individually, but since the titers were similar for allmice in the group, the antisera individual mice in each group werepooled and all further experiments were done with the pooled antisera.ELISA plates were prepared by diluting each PS-BSA conjugate 1:200 inPBS and adding 100 μl per well to a 96-well microtiter plate (ImmulonIIHB). The plates were stored overnight at 4° C. before use. The plateswere washed with PBS buffer 5 times and blocked with PBS buffercontaining 1% (w/v) of BSA (blocking buffer) for 1 hr at ambienttemperature. The antisera were added in blocking buffer at 1:100dilution, followed by serial 3-fold dilutions. After overnightincubation at 4° C., the plates were washed with PBS buffer 5 times andrabbit anti-mouse-alkaline phosphatase conjugate antibody (Zymed)diluted 1:3000 in blocking buffer was added. After incubating anadditional hour, the plates were washed (5×) with PBS buffer and thebound antibody was detected by adding 1 mg/ml p-nitrophenyl phosphatesubstrate (Sigma-Aldrich) in 50 mM sodium carbonate buffer, pH 9,containing 1 mM MgCl₂. The absorbance at 405 nm after 1 hr incubation atambient temperature was measured using a BioRad Model 550 microtiterplate reader. Antisera were tested against the homologous antigen PS-BSAconjugate and against DeNAc-BSA.

FIG. 10 (upper panel) shows the titers for each group of pooled antiseraagainst the homologous antigens and DeNAc PSA after each immunization asmeasured by ELISA. The titers for homologous antigens varied widely. Forall vaccines, the titer elicited by the 10 μg dose was higher than thatelicited by the 2 μg dose but did not increase after the second dose.The relative titers against homologous antigens were consistent for bothdosages and for all post primary immunizations. The order of decreasingimmunogenicity for homologous antigens was NPrSia>DeNAc>OS>TcAc, NPr.The TcAc-TT and NPr-TT vaccines elicited very low titers against thehomologous antigens that did not increase after booster doses (FIG. 10).

The reactivity of the pooled antisera from each dose for the DeNAc-BSAantigen was also evaluated by ELISA. All of the PS-TT vaccines containedsome fraction of Neu residues (Table 2) and all five vaccines elicitedtiters greater than >10,000 against DeNAc-BSA (FIG. 10, lower panel)that did not increase after the second immunization (FIG. 10). Eventhough the amount of Neu in each PS-TT vaccine and in each dose variedover a wide range (from ˜0.3 μg/dose to ˜10 μg/dose), all vaccines atboth doses elicited anti-DeNAc titers of roughly the same magnitude.None of the antisera was reactive against unmodified PSA by ELISA (titer<1:50). The result suggests that the zwitterionic Neu component of allof the antigens is immunogenic and is the immunodominant determinant ofthe PS-TT vaccines.

Example 15 Evaluating Binding of PS-TT Antisera to NeisseriaMenengitidis Group B (NmB) Bacteria

The ability of the PS-TT vaccines to elicit antibodies that bind to NmBwas tested by flow cytometry. The NmB strain NMB was grown to an O.D.₆₂₀of 0.6 in Mueller-Hinton media containing 0.3% glucose. The cells werepelleted, washed, and resuspended in 80% of the original volume inblocking buffer. The resuspended bacteria were added to the reactionmixture such that the final concentrations were 50% resuspendedbacteria, 10% antiserum, and 40% blocking buffer. The mixture wasincubated at 4° C. for 2 hr with periodic gentle agitation. The cellswere pelleted and resuspended in 100 μl of a 1:300 dilution in blockingbuffer of fluorescein isothiocyanate (FITC)-conjugated goat anti-mousesecondary antibodies. FITC-conjugated antibodies against IgG(H+L)F(ab′)₂ and IgM (Jackson ImmunoResearch, West Grove, Pa.) as well asIgG1, IgG2a, IgG2b, and IgG3 (Bethyl Laboratories, Montgomery, Tex.)were used. After the secondary antibody was added, the tubes wereincubated for 1 hr at 4° C. with periodic gentle agitation. The cellswere pelleted and resuspended in 450 μl of PBS containing 0.5%formaldehyde (weight/volume), freshly made and filtered. The sampleswere immediately analyzed by flow cytometry (BD FACSCalibur System, BDBiosciences, San Jose, Calf.). As shown in FIG. 11A, all of the PS-TTvaccines except for the OS-TT 2 μg dose elicited both IgG and IgMantibodies after the third dose that bound to strain NMB. Althoughbinding appeared to be relatively poor in some cases, binding by the“paradigm” mAbs SEAM 2 and 3 is also poor compared to the autoreactivemAb SEAM 12, which binds to the bacteria very strongly (FIG. 11A). Thereasons for the apparently complex binding characteristics of two mAbs(SEAM 2 and 3) that are nonetheless protective have to do with thedistinctive characteristics of the antigens recognized by the mAbs. BothSEAM 2 and 3 apparently recognize antigens that are non-capsular and areneither highly nor uniformly expressed over the entire surface of thebacteria. Immunization with the carrier tetanus toxoid protein alonealso elicited polyreactive IgM but not IgG antibodies that could bind toNMB (FIG. 11A). In general, the 10 pg dose PS-TT antisera bound morestrongly to the bacteria than the 2 μg dose antisera. The exception wasNPrSia-TT, in which the pattern was reversed. IgG binding was somewhatstronger in the 10 μg dose antisera than the 2 μg, and the differencewas even more pronounced with IgM.

Some of the PS-TT antisera were further analyzed to determine which IgGsubclasses bound to the bacteria. Representative data obtained withDeNAc-TT antisera is shown in FIG. 11B. With the exception of IgG1, allthe antisera contained antibodies of all IgG subtypes that bound to thebacteria. DeNAc-TT (shown) was the only antiserum that contained IgG1Ab. The amounts of bound IgG2a, IgG2b, and IgG3 were roughly the same.Since the antisera were all pooled samples of 5 mice each, it ispossible that there could have been individual differences in theprevalence of IgG2a, IgG2b, and IgG3 Ab that were obscured by thepooling. The above results demonstrate that all of the PS-TT vaccineselicited anti-Neu-containing PSA antibodies and all of them werereactive with group B strain NMB.

Example 16 Evaluating Functional Activity of PS-TT Antisera withNeisseria meningitidis Group B, C, X, Y, W135 Bacteria

Activation of complement protein deposition. The ability of antibodieselicited by immunization with PS-TT conjugate vaccine antisera toactivate deposition of human complement components on Neisseriameningitidis groups B, C, X, Y, and W135 bacteria was determined asdescribed in Example 10. The results are shown in FIG. 12.

Deposition of complement components on the cell surface increases thefluorescence of the cells and is indicated by the shift in fluorescencepeak to the right of the graph. The antibody activation is indicated bythe lack of fluorescence of cells alone with active complement orantisera with heat-inactivated complement.

The results demonstrate that all the antisera strongly activatedcomplement deposition on NmB, and there was little difference in theamount of complement deposited between the antisera elicited bydifferent antigens or 2 μg and 10 μg doses (data not shown). Theconsistency of complement activation is in accordance with thesimilarity seen in the anti-deNAc titers. It suggests that all theantigens were equally effective, and increasing the dose of PS did notincrease the anti-Neu PSA antibody responses of the vaccines.

Recently, we discovered that SEAM 2, 3 and 18 (Granoff et al Id.)antibodies are reactive with and have functional activity againstmeningococcal strains from serogroups A, C, W135 as well as B (Flitter,BA and Moe, GR, unpublished). Therefore, we also measured the ability ofthe antisera to activate complement protein deposition on strainsrepresentative of all N. meningitidis serogroups. At least one of theantisera pools was able to activate complement protein deposition ongroup A, B, C, X, Y, or W135 strains. In particular, the 2 μg or 10 μgantisera pools activated complement protein deposition on group A, B, C,X and Y strains but not on W135 strains. Only the NPrSia antisera poolsshowed activity against group W135 strains. The results from the controltetanus toxoid only antisera were uninterpretable since the antisera hada very high background signal with heat inactivated complement,particularly with group C, Y and W135 strains. In contrast, all of thecomplement activation activity observed with the PS-TT conjugate vaccineelicted antisera was dependent on active complement.

The results suggest that all Neisseria meningitidis strains regardlessof capsular group either express or acquire exogenously poly alpha (2→8)PSA antigens likely containing Neu and that vaccines elicitinganti-NeuPSA antibodies may be protective against disease caused by allmeningococcal group strains, as activation of complement factordeposition on the cell surface of N. meningitidis bacteria is correlatedwith protection against disease (Welsch et al, J Infec Dis, 2003,188:1730).

Serum bactericidal activity. Complement-mediated bactericidal activitywas measured with N. meningitidis group B strain NMB and group C strain4243 as follows. After overnight growth on chocolate agar (Remel),several colonies of N. meningitidis were inoculated into inMueller-Hinton broth (starting A_(620 nm) of ˜0.1) and the test organismwas grown for approximately 2 hrs to an A_(620 nm) of ˜0.6. Afterwashing the bacteria twice in D-BSA approximately 300 to 400 CFU wereadded to the reaction mixture. The assays were performed with humancomplement from a donor that lacks bactericidal activity against thetest strain in the absence of added antibody up to 40% complement. Thefinal reaction mixture of 40 PL contained 20% (v/v) complement, antiseradiluted in D-BSA buffer. CFU/ml in the reaction mixtures were determinedafter overnight growth on chocolate agar (Remel). Bactericidal titers orconcentrations were defined as the serum dilution resulting in a 50%decrease in colony forming units (CFU) per ml after 60 minutesincubation of bacteria in the reaction mixture, compared to the controlCFU per ml at time 0. Typically, bacteria incubated with the negativecontrol antibody and complement showed a 150 to 200% increase in CFU/mlduring the 60 minutes of incubation.

Although the PS-TT antisera were able to activate complement proteindeposition on group B bacteria, none of the antisera were able tomediate bacteriolysis in the presence of complement. The mechanisticreasons for this are unknown, but similar functional characteristics areobserved for the protective, non-autoreactive mAb SEAM 3 (Granoff etal., J. Immunol, 1998, 160: 5028; Moe et al., Infect. Immun. 2005,73:2123). In contrast, the antisera pools for NPr, DeNAc, and TcAc (all2 μg dose post 3^(rd) injection) exhibited high titers against the groupC strain showing that antibodies elicited by the vaccines can mediatecomplement-dependent bacteriolysis, which is the hallmark of protectionagainst meningococcal disease (Goldschneider et al, J. Exp. Med., 1969,129:1327).

TABLE 3 Serum bactericidal activity of PS-TT antisera pools against NmCstrain 4243 with human complement. 1/Titer Antisera (2 μg dose) Expt. 1Expt. 2 Expt. 3 TT alone <8 <8 <16 OS-TT <8 <8 <16 NPrSia-TT <8 <8 <16NPr-TT >256 >256 2048 DeNAc-TT 256 96 8192 TcAc-TT >256 128 6144

Passive protection in infant rats. Infant (4-6 days) Wistar rats weretaken from the mothers and randomly divided into groups of 5 rats each.Each pup was given 100 μl of antiserum diluted 1:10 in sterile PBScontaining 1% BSA (PBS-BSA) intraperitoneally and then returned to theirmothers while the challenge bacteria were prepared. NmB strain M986 orNmC strain 4243 was grown to O.D.₆₂₀ 0.6 in Mueller-Hinton broth with0.3% glucose, washed, resuspended in PBS-BSA, and diluted to 10⁴ CFU/ml.Each rat pup was given 100 μl of bacteria, so the final challenge dosewas ˜1000 CFU/rat. The pups were returned to their mothers. The nextday, the pups were anesthetized with isoflurane and blood was obtainedby cardiac puncture using a heparanized needle. The animals wereeuthanized by CO₂ anoxia, and 100 μl, 10 μl, and 1 μl of the blood wasplated on chocolate agar (Remel). The plates were incubated at 37° C.,5% CO₂ overnight then the colonies were counted.

Some of the PS-TT antisera were protective or partially protectiveagainst NmB or NmC bacteremia in the infant rat model of passiveprotection (FIG. 13). Protection was calculated by comparing thegeometric mean CFU/ml of the vaccine-elicited antisera to the geometricmean CFU/ml of the antisera from mice immunized with the TT carrierprotein alone. Four of the PS-TT antisera pools provided passiveprotection against strain M986 that was different from the TT antiserum.The antisera included NPrSia-TT 10 μg, DeNAc-TT 2 μg (p<0.05), NPr-TT 2μg, and OS-TT 2 μg (p<0.01).

The 2 μg doses tended to be more protective than the 10 μg doses,although the difference was not quite significant (p=0.052). If theNPrSia-TT antiserum, which shows the opposite pattern, is removed fromthe analysis, the 2 μg doses were significantly more protective than the10 μg doses (p=0.013). NPrSia-TT may be an exceptional antiserum. It isthe only one in which the 10 μg doses bound more strongly to thebacteria than the 2 μg, and the NPrSia-TT vaccine formulation containedby far the least amount of neuraminic acid. It is possible that theNPrSia-TT 2 μg dose formulation simply did not contain enough neuraminicacid residues to elicit strong binding and protective antibodies. Forthe other PS-TT vaccines, the amount of Neu in the 2 μg dose appears tobe sufficient to elicit the maximum anti-NeuPS antibody responses andthe 10 μg dose did not convey any additional benefit.

Passive protection in an ex vivo human blood model of meningococcalbacteremia. As note above in Example 10, an alternative approach toevaluating the ability of antibodies elicited by a vaccine to protectagainst disease caused by NmB is to determine whether the antisera canlyse or inhibit the growth of NmB ex vivo in human blood. Antisera(pooled antisera from CD1 mice immunized with 10 μg of PS -TT conjugatevaccines as described in Example 15) and the test bacteria(approximately 1000 CFU of NmB strain NZ98/254 freshly grown inMuller-Hinton media as described in Example 15 above) were combined infreshly obtained human blood from a donor who lacks antibodies to thetest strain in sterile glass vials and prepared and tested as describedin Example 15. The results of testing the antisera in the ex vivo humanblood model of meningococcal bacteremia are shown graphically in FIG.14, and are similar to the results of Example 15. Thus, antibodieselicited by the PS-TT vaccines described herein support application ofthe vaccines for protection against disease caused by N. meningitidisbacteria expressing a de-N-acetylated sialic acid (deNAc SA) epitope.

Example 17 Binding of Vaccine Elicited Antibodies to PSA DerivativesExpressed by the Jurkat T-Cell Leukemia Cell Line

To measure binding of antibodies elicited by the PS-TT conjugatevaccines prepared in Example 13, Jurkat cells were tested as describedin Example 11 except that after blocking, pooled antisera from CD1 miceimmunized with the 3 doses of 2 μg of total sialic acid PS-TT conjugatevaccine was added and incubated overnight at 4° C. The results of theJurkat cell binding experiment are shown in FIG. 15. Although there is asmall amount of non-specific binding of the TT negative control antiseracompared to the cells only, all of the antisera pools showed strongbinding to Jurkat cells as indicated by the increase in fluorescence ofthe gated cells. Thus, the results confirm that the PS-TT conjugatevaccines elicit antibodies that are reactive with neuraminicacid-containing antigens expressed by Jurkat T-cell leukemia cells.

Example 18 Activation of Complement Deposition by Vaccine ElecitedAntibodies on Jurkat T-Cell Leukemia Cells, SK-MEL 28 Melanoma, andCHP-134 Neuroblastoma Cells

Activation of complement-mediated cytotoxicity is an important mechanismfor antibody dependent killing of cancer cells (Maloney et al, SeminOncol, 2002. 29(1 Suppl 2):2). Therefore, the ability of PS-TT antiserato activate deposition of human complement proteins on CHP-134neuroblastoma, Jurkat T-cell leukemia, and SK-MEL 28 melanoma cells wasmeasured by flow cytometry.

Cells (approximately 10⁵ per well) were plated onto a flat bottom96-well tissue culture plate (Nunc) and incubated with growth mediumovernight before assay. Cells were detached from the plate (Jurkat cellsare non-adherent) by either trypsin (SK-MEL-28) or Cell DispersalReagent (CDR, Guava Technologies) (CHP-134) before being collected intoa 96-round bottom plate (Falcon), spun at 1000×g for 5 minutes, thesupernatant was removed and the cells were resuspended in a 1:10dilution of the PS-TT or TT (negative control) antisera (2 μg dose) orno antisera in 95 μl of standard cell culture medium (RPMI-1640 growthmedium supplemented with 10% fet al bovine serum). Human complement (5μl) from a donor with no intrinsic activity against the cancer celllines was added and mixed. After 30 minutes at ambient temperature, thecells were pelleted by centrifugation (above), washed with PBS bufferand suspended in PBS buffer containing a 1:100 dilution ofFITC-conjugated sheep anti-human C3c antibody (BioDesign International).After 30 minutes incubation at ambient temperature, the cells werepelleted and washed as before and finally resuspended in PBS buffercontaining 1% formaldehyde. The relative fluorescence of the cells wasusing a Guava EastCyte flow cytometer (Guava Technologies). Controlsamples which contained no antisera, were used to establish baselinefluorescence.

All of the PS-TT test antisera, but not the control TT only antisera,was able to activate complement protein deposition on all three celllines (FIG. 16).

Example 19 Effect of Vaccine Elicited Antibodies on the Viability ofJurkat T-Cell Leukemia Cells

The effect of PS-TT antisera on the viability of the human T-cellleukemia Jurkat cell line in culture was measured using a cell viabilityassay. Cells were incubated with a 1:20 dilution of the antisera for 24hours. Jurkat cells were incubated at a concentration of 2×10⁵ cells/mlin round-bottom 96-well plates (Falcon), 200 μl/well. Plates were thenspun at 1,000×g for 5 minutes. The cells were resuspended in GuavaViaCount reagent and read on a Guava EasyCyte flow cytometer, using theGuava ViaCount assay (all from Guava Technologies).

As shown in FIG. 17, antibodies elicited by the NPrSia-TT and TcAc-TTvaccines were able to reduce the viability of Jurkat cells.

Example 20 Immunohistochemical Analysis of TCAC-TT Vaccine ElicitedAntibodies Binding to Antigens Expressed in Primary Human Cancers

Cancer cell lines are clonal but can undergo changes when passaged manytimes in cell culture. Therefore, it is important to demonstrate thatantigens recognized by antibodies elicited by the PS-TT vaccines arealso present in primary human tumors. Also, for an immunotherapeuticapproach to be useful, it is important that the antigens targeted areeither not expressed or expressed at greatly reduced levels in normaltissues. Since the TcAc-TT antisera exhibited the greatest overallfunctional activity against meningococcal bacteria and Jurkat cells, thereactivity of the antisera with normal and cancerous tissues wasevaluated by immunohistochemistry (IHC).

Frozen, unfixed tissue arrays containing 28 normal and cancer tissuesincluding normal brain, breast, colon, skeletal muscle, kidney, liver,lung pancreas, prostate, skin, small intestine, stomach, ovary, anduterus and malignant tumors from the same tissues were obtained fromBioChain Institute, Inc. (Hayward, Calif.). The slides were rinsed withPBS buffer then briefly washed with cold (−20° C.) acetone. Endogenousperoxidases were blocked by incubation with PEROXIDAZED 1 (BiocareMedical, Concord, Calif.) followed by washing with PBS buffer andblocked with 2.5% (volume/volume) normal horse serum (Vector Labs,Burlingame, Calif.) for 30 min. TT control and TcAc-TT antisera (2 μgdose) diluted in DA VINCI GREEN (Biocare Medical) were added and theslides incubated in a humid chamber overnight at 4° C. Unbound antibodywas removed by buffer rinses. Bound antibody was then detected using AECsubstrate (Vector Labs) following the manufacturer's directions. Afteradditional washes, nuclei were counterstained using Mayer's hematoxylinQS (Vector). Finally, the slides were mounted in aqueous mounting medium(VECTRAMOUNT™ AQ, Vector) and viewed using a Zeiss Axioplan microscope.

The TT and TcAc-TT antisera showed no or weak staining to the normaltissues and the TT antisera was not reactive with any of the tumortissues. However, TcAc-TT antisera showed clear staining of skinmelanoma metastasized to brain and adenocarcinomas of the pancreas,stomach, ovary, and uterus, and a renal cell carcinoma. An examplecomparing the TT and TcAc-TT staining of normal ovary and ovarianadenocarinoma is shown in FIG. 18. The results show that the antigensreactive with antibodies elicited by the TcAc-TT vaccine are expressedin only or at higher levels in several primary human tumors but not innormal human tissues.

Example 21 Production of Monoclonal Antibodies Using the DeNAc-TTConjugate Vaccine

Four to six weeks old female CD1 mice were immunized with the DeNAc-TTvaccine as described in Example 14. Three days after the 3^(rd) dose,mice were sacrificed and their spleen cells were fused with myelomacells P3X63-Ag8.653 at a ratio of 5 spleen cells to 1 myeloma cells.After two weeks incubation in HAT selective medium, hybridomasupernatants were screened for antibody binding activity by ELISA,performed on microtiter plates coated with the DeNAc-BSA derivative(Example 9). A large number of positive wells (250) were identified.Cell culture supernatants from the DeNAc-BSA positive wells were thensubjected to a second screen based on the ability of the antibody in thesupernatant to activate complement deposition on Jurkat cells asdescribed in Example 18. Of the original 250 DeNAc-BSA positive wells 11were also positive for complement activation. Five (5) of the 11hybridomas were cloned twice by limiting dilution and then expanded andfrozen for subsequent use in tissue culture.

The subclasses of the monoclonal antibodies were determined using amouse monoclonal antibody isotyping kit (Southern Biotech, Birmingham,Ala.). Among the selected mAbs, one IgM anti-DeNAc mAb, designated DA2,was used in all of the binding and functional studies described below.This monoclonal antibody was purified from tissue culture by ammoniumsulfate precipitation and size exclusion chromatography (ToyoPearlHW-55F, Sigma-Aldrich) in buffer containing 2 mM arginine, 0.002% Tween20, 24 mM sucrose, pH 7 (all from Sigma-Aldrich). The IgM-containingfractions were combined, sterile filtered, lyophilized, and stored at−80° C. until used. The lyophilized mAb was resuspended in 1/10^(th) theoriginal volume of sterile water for use in the experiments describedbelow in Example 23. The DA2 mAB was found to be highly specific for anynon-reducing end neuraminic acid residue, regardless of the adjacentresidue or glycosidic linkage (data not shown).

Example 22 Cloning and Sequencing of Nucleic Acid Encoding the DA2 MAb

To investigate the molecular basis for antigen recognition, the variableregion (V) gene of the DA2 murine mAb was cloned and sequenced asfollows.

The variable region gene of the immunoglobulin heavy and light chainsfrom a DA2-expressing mouse hybridoma cell line was amplified by PCRusing degenerate primers and cloned using the TOPO TA Cloning® Kit(Invitrogen, Carlsbad, Calif.) (Invitrogen,) as described by Wang et al.(2000) J Immunol Methods 233, 167-77 using E. coli strain XL-2 Blue as ahost. Plasmid DNA from individual transformants selected onLB-ampicillin plates was isolated using the Qiagen Mini Prep Kit(Qiagen) according to the manufacturer's instructions. The cloned V genefrom three clones was sequenced by Davis Sequencing (Davis, Calif.).

The mAb nucleotide sequence of DA2 was analyzed using IGMT/V-QUEST andthe mouse immunoglobulin nucleotide sequence data-base through theonline web facilities of the international ImMunoGeneTics® informationsystem (IMGT, on the internet at imgt.cines.fr) that was initiated andcoordinated by Marie-Paule Lefranc (Université Montpellier II, CNRS,LIGM, IGH, IFR3, Montpellier, France).

The nucleic acid and amino acid sequences of the variable regions of theDA2 heavy chain polypeptide and light chain polypeptide are provided inFIGS. 19 and 20 with the framework (denoted by, e.g., FR1-IMTG) and CDRregions indicated as defined by the International ImmunogeneticsInformation System (IMGT) definitions (Lefranc et al. IMGT, theinternational ImMunoGeneTics information system®. Nucl. Acids Res.,2005, 33, D593-D597).

Example 23 Effect of mAb DA2 on the Viability of Human Melanoma CellLine SK-MEL 28

To determine the effect of DA2 (1, 0. 5, and 0.25 μg/ml) and anirrelevant IgM (Southern Biotech) control mAb (5 μg/ml) on cellviability, the mAbs were incubated in centuplicate with SK-MEL 28 cellsfor 48 hrs. The cells were then analyzed by flow cytometry. Cellviability was determined using ViaCount Reagent (Guava Technologies), asper manufacturer's instructions. Briefly, cells were cleaved from thetissue culture plate, collected by centrifugation and resuspended inViaCount Reagent. Cell viability was analyzed using a program on theGuava EasyCyte flow cytometer that had preset gates for live, apoptotic,and dead cells. DA2 was found to reduce the number of viable cells, andincreases the number of apoptotic and dead cells compared to theirrelevant control mAb at all concentrations tested (FIG. 21).In datanot shown, DA2 can inhibit the growth Neisseria meningtidis strains fromserogroups A, B, C, X, Y, and W135.

Thus, SEAM 3 and DA2 which have different fine antigenic specificitiesbut recognize in common PS antigens containing Neu at the non-reducingend both have functional activity against Neisseria meningitidis andcancer cells that express Neu-containing sialic acid antigensetc.]

It was also found that OS derivatives made from PSA alpha (2→9) capsularmaterial of N. meningitidis serogroup C contain the immunodominantNeu-epitope recognized by the DA2 monoclonal antibody. As such, the dataindicate that a new class of OS alpha (2→9) derivative vaccines(including mixtures of alpha (2→8) and (2→9) glycosidic linkages) can beapplied in a similar manner as described for OS alpha (2→8) derivatives.

The above results demonstrate that the shortest oligosialic acid oroligosaccharide (OS) contained a mixture of degree of polymerization(Dp) of 2 to 6, but mostly 4 to 6, and that a tetramer exhibits all ofthe activity of the much longer derivatives. The results alsodemonstrate that a vaccine composed of OS derivative with these featuresis highly immunogenic and elicits an antigen-specific antibody responsethat (i) activates protective mechanisms present in human blood(complement mediated bacteriolysis and/or opsonophagocytosis) thatdecreases viability of the bacteria in human blood, and (ii) is reactivewith neuraminic acid-containing antigens expressed by cancer cells.

In addition, methods have been described for producing andcharacterizing defined PS-TT vaccines including DeNAc-TT, NPrSia-TT,TcAc-TT and OS-TT. Extensive characterization of antisera against thePS-TT vaccines further supports the finding that the smallest OS vaccinederivatives bearing a non-reducing end de-N-acetyl sialic acid residuecontains the minimal features necessary for effective vaccine activity.Surprisingly, while the TcAc-TT vaccine elicited very low titers againstthe homologous antigen, antisera against TcAc-TT exhibited a broadspectrum of activity against both Neisseria meningitidis serogroups andcancer cells. Moreover, it was found that the NPrSia-TT, TcAc-TT andOS-TT conjugate vaccines as well as the unconjugated antigens having adegree of polymerization (dp) of about 2-20, particularly a dp of about2-10 or less, exhibited an IC50 of less than about 0.1 μg/ml forinhibiting binding SEAM 2 (TcAc) or SEAM 3 (NPrSia-TT and OS-TT) to NPror dodecylamine-NPr, further illustrating that the OS vaccinederivatives bearing a non-reducing end de-N-acetyl sialic acid residuecontains the minimal features necessary for effective vaccine activity.

It was also found that in addition to OS derivatives produced from PSAalpha (2→8) precursor material (such as obtainable from N. meningitidisserogroup B or E. Coli K1), OS derivatives derivable from PSA alpha(2→9) material (such as from N. meningitidis serogroup C) exhibitsimilar properties, supporting a new class of PS-TT vaccines comprisingalpha (2→9), or a mixture of alpha (2→8) and alpha (2→9), glycosidiclinkages. For example, capsular polysaccharide isolated from E. coli K92strains (Devi et al. Proc. Natl. Acad. Sci. USA, 1991, 88:7175). PSvaccines containing alpha (2→9) glycosidic linkages can have particularadvantages for application against N. meningitidis serogroup C.

The results also demonstrate that while the PS-TT vaccines have a rangeof different activities, the non-reducing end de-N-acetyl neuraminicacid residue component present in the OS derivatives (i) is found in allof the PS-TT antigens, (ii) is immunogenic and the immunodominantdeterminant of the PS-TT vaccines, and (iii) PS-TT antisera for allvaccines were able to activate complement factor deposition on the cellsurface of Neisseria meningitidis groups B, C, X, Y, and W135, which isa known correlate of protection against disease caused by thesebacteria. Moreover, all of the PS-TT antisera were able to activatecomplement protein deposition on different cancer cells, and it wasdemonstrated that reactive antigens were expressed in only or at higherlevels in several primary human tumors but not in normal human tissues.Lastly, a non-reducing end Neu-specific monoclonal antibody DA2 wasisolated, sequenced, propagated, and found to bind with higher affinityto the immunodominant epitope than any of the SEAM antibodies, as wellas reduce the number of viable cancer cells, and increases the number ofapoptotic and dead cells compared to the irrelevant control mAb at allconcentrations tested.

It is evident from the above results and discussion that OS derivativesand vaccines can be produced to exhibit protective effects againstdisease caused by N. meningitidis,particularly serogroups B and C. Italso is evident that the OS derivatives and antibodies generatedthereto, including DA2, are applicable for detecting a cancerous cell ina subject, inhibiting growth of a cancerous cell in a subject, elicitingantibodies in a subject, eliciting antibodies to a cancerous cell, andthe like. As such, the compositions and methods disclosed herein finduse in a variety of different applications and represents a significantcontribution to the art.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. A method of producing an isolated alpha (2→8) or (2→9) oligosialicacid derivative, said method comprising: generating an alpha (2→8) or(2→9) oligosialic acid derivative having one or more de-N-acetylatedresidues by treating an alpha (2→8) or (2→9) oligosialic acid precursorhaving a reducing end and a non-reducing end with sodium borohydrideunder conditions for de-N-acetylating said non-reducing end; andisolating the alpha (2→8) or (2→9) oligosialic acid derivative having(i) a degree of polymerization of about 2-20, and (ii) one or morede-N-acetylated residues and a non-reducing end that is resistant todegradation by exoneuraminidase, whereby said isolated alpha (2→8) or(2→9) oligosialic acid derivative is produced.
 2. The method of claim 1,wherein said non-reducing end of said oligosialic acid derivative is ade-N-acetylated residue.
 3. The method of claim 2, wherein saidde-N-acetylated residue is neuraminic acid.
 4. The method of claim 1,wherein said oligosialic acid derivative comprises one or more N-acylgroups other than N-acetyl.
 5. The method of claim 1, wherein saidN-acyl group is trichloroacetyl.
 6. The method of claim 1, wherein saidoligosialic acid precursor is obtainable from acid hydrolysis of apolysialic acid polymer obtainable from a bacterium selected from thegroup consisting of E. coli K1, Neisseria meningitidis serogroup B, andNeisseria meningitidis serogroup C.
 7. The method of claim 1, whereinsaid oligosialic acid derivative has a degree of polymerization rangingfrom 2 to
 10. 8. The method of claim 1, wherein said oligosialic acidderivative is comprised as an isolated mixture of alpha (2→8) or (2→9)oligosialic acid chains.
 9. The method of claim 8, wherein said mixtureof alpha (2→8) or (2→9) oligosialic acid chains comprises shorter lengthchains and a ratio of sialic acid to de-N-acetylated sialic acid of 3:1.10. The method of claim 8, wherein said mixture of alpha (2→8) or (2→9)oligosialic acid chains comprises longer length chains and a ratio ofsialic acid to de-N-acetylated sialic acid of and 10:1
 11. The method ofclaim 1, which further comprises conjugating a second molecule to saidisolated alpha (2→8) or (2→9) oligosialic acid derivative, wherein saidsecond molecule is selected from the group consisting of protectinggroup, amino acid, peptide, polypeptide, lipid, carbohydrate, nucleicacid and detectable label.
 12. The method of claim 11, wherein saidsecond molecule is an immunomodulator.
 13. The method of claim 12,wherein said immunomodulator is a toxin or derivative thereof.
 14. Themethod of claim 13, wherein said toxin or derivative thereof is tetanustoxoid.
 15. The method of claim 1, wherein said isolated oligosialicacid derivative is capable of inhibiting SEAM 2, SEAM 3, or DA2 bindingto dodecylamine N-propionyl NmB polysialic acid or N-propionyl NmBpolysialic acid at an IC50 of less than about 0.1 μg/ml.
 16. The methodof claim 1, which further comprises enriching for alpha (2→8)oligosialic acid derivative having a non-reducing end that is resistantto degradation by exoneuraminidase by exposure of said alpha (2→8) or(2→9) oligosialic acid derivative to exoneuraminidase.
 17. An isolatedalpha (2→8) or (2→9) oligosialic acid derivative produced according tothe method of claim
 1. 18. A composition comprising an isolated alpha(2→8) or (2→9) oligosialic acid derivative produced according to themethod of claim 1, wherein said isolated alpha (2→8) or (2→9)oligosialic acid derivative comprises as mixture of oligosialic acidderivatives of variable chain lengths each having a non-reducing endde-N-acetyl residue.
 19. A composition comprising an alpha (2→8) or(2→9) oligosialic acid derivative having a degree of polymerization ofabout 2-20, and a reducing end and a non-reducing end, wherein saidnon-reducing end comprises a de-N-acetylated residue that is resistantto degradation by exoneuraminidase.
 20. The composition of claim 19,wherein said non-reducing end of said oligosialic acid derivative is ade-N-acetylated residue.
 21. The composition of claim 19, wherein saidde-N-acetylated residue is neuraminic acid.
 22. The composition of claim19, wherein said oligosialic acid derivative comprises one or moreN-acyl groups other than N-acetyl.
 23. The composition of claim 19,wherein said reducing end of said isolated oligosialic acid derivativeis reduced.
 24. The composition of claim 19, wherein said oligosialicacid is obtainable from a polysialic acid polymer obtainable from abacterium selected from the group consisting of E. coli K1, Neisseriameningitidis serogroup B, and Neisseria meningitidis serogroup C. 25.The composition of claim 24, wherein said oligosialic acid derivativecomprises a degree of polymerization ranging from about 2 to
 10. 26. Thecomposition of claim 19, wherein said oligosialic acid derivative iscomprised as an isolated mixture of oligosialic acid chains.
 27. Thecomposition of claim 26, wherein said mixture of oligosialic acid chainscomprises shorter length chains and a ratio of sialic acid tode-N-acetylated sialic acid of about 3:1.
 28. The composition of claim26, wherein said mixture of oligosialic acid chains comprises longerlength chains and a ratio of sialic acid to de-N-acetylated sialic acidof about 10:1.
 29. The composition of claim 19, wherein said oligosialicacid derivative comprises a conjugate.
 30. The composition of claim 29,wherein said conjugate comprises as a first molecule said oligosialicacid derivative conjugated to one or more second molecules selected fromthe group consisting of protecting group, amino acid, peptide,polypeptide, lipid, carbohydrate, nucleic acid and detectable label. 31.The composition of claim 30, wherein said second molecule is animmunomodulator.
 32. The composition of claim 31, wherein saidimmunomodulator is a toxin or derivative thereof.
 33. The composition ofclaim 32, wherein said toxin or derivative thereof is tetanus toxoid.34. The composition of claim 19, wherein said oligosialic acidderivative is comprised as a formulation containing one or moreimmunogenic excipients.
 35. The composition of claim 19, wherein saidoligosialic acid derivative is capable of inhibiting SEAM 2, SEAM 3 andDA2 binding to dodecylamine N-propionyl NmB polysialic acid orN-propionyl NmB polysialic acid at an IC50 of less than about 0.1 μg/ml.36. The composition of claim 19, wherein said isolated alpha (2→8) or(2→9) oligosialic acid derivative comprises as mixture of oligosialicacid derivatives of variable chain lengths that have a non-reducing endenriched with de-N-acetyl residues.
 37. An isolated antibody specificfor an alpha (2→8) or (2→9) oligosialic acid derivative that comprises anon-reducing end enriched for one or more de-N-acetylated residues andis resistant to degradation by exoneuraminidase.
 38. The isolatedantibody of claim 37, wherein said antibody is capable of complementmediated bacteriolysis and opsonophagocytosis of Neisseria meningitidisgroup B (NmB) and group C (NmC) bacteria.
 39. The isolated antibody ofclaim 37, wherein said antibody is specific for non-reducing endde-N-acetyl sialic acid residue.
 40. The isolated antibody of claim 39,wherein said antibody is a monoclonal antibody having a light and heavychain variable complementarity determining region polypeptide sequenceas depicted in FIGS. 19 and
 20. 41. The isolated antibody of claim 37,wherein said antibody is a monoclonal antibody having a complementaritydetermining region (CDR) polypeptide sequence selected from a CDRpolypeptide sequence depicted in FIG. 19 or
 20. 42. The isolatedantibody of claim 41, wherein said monoclonal antibody is a humanizedmonoclonal antibody.
 43. A method of detecting a cancerous cell in asubject, the method comprising contacting a biological sample obtainedfrom a subject suspected of having cancer with an antibody according toclaim 37, wherein the binding of the antibody is indicative of thepresence of cancerous cells in the subject.
 44. A method of inhibitinggrowth of a cancerous cell in a subject, said method comprising:administering to the subject an effective amount of a pharmaceuticallyacceptable formulation comprising an antibody according to claim 37,wherein said administering facilitates reduction in viability ofcancerous cells exposed to said antibody.
 45. A method of elicitingantibodies in a subject, where the antibodies specifically bind abacterium comprising a de-N-acetylated sialic acid (deNAc SA) epitope,the method comprising: administering to a subject an immunogeniccomposition comprising an alpha (2→8) or (2→9) oligosialic acidderivative having a degree of polymerization of about 2 to 20, and areducing end and a non-reducing end, wherein said non-reducing end isenriched for one or more de-N-acetylated residues and resistant todegradation by exoneuraminidase, and wherein said administering iseffective to elicit production of an antibody that specifically binds adeNAc SA epitope of a bacteria.
 46. The method of claim 45, wherein thebacteria is Neisseria meningitidis group B, Neisseria meningitidis groupC, or Escherichia coli K1.
 47. A method of eliciting antibodies to acancerous cell comprising a de-N-acetylated sialic acid (deNAc SA)epitope in a subject, the method comprising: administering to a subjectan immunogenic composition comprising an alpha (2→8) or (2→9)oligosialic acid derivative having a degree of polymerization of about 2to 20, and a reducing end and a non-reducing end, wherein saidnon-reducing end is enriched for one or more de-N-acetylated residuesand resistant to degradation by exoneuraminidase, and wherein saidadministering is effective to elicit production of an antibody thatspecifically binds a deNAc SA epitope of said cancerous cell.
 48. Themethod of claim 47, wherein the cancer is a melanoma, lymphoma, orneuroblastoma.
 49. The method of claim 47, wherein the alpha (2→8) or(2→9) oligosialic acid derivative of the immunogenic composition isprepared by selective de-acetylation of non-reducing end residue bysodium borohydride reduction.
 50. The composition of claim 19, whereinsaid isolated alpha (2→8) or (2→9) oligosialic acid derivative comprisesan aggregate of the polysialic acid derivative.
 51. The composition ofclaim 50, wherein said aggregate comprises a microscopic particle.
 52. Amethod of producing an aggregate comprising an alpha (2→8) or (2→9)oligosialic acid derivative, the method comprising: admixing one or morealpha (2→8) or (2→9) oligosialic acid derivatives under aggregatingconditions so as to form an aggregate.
 53. The method of claim 52,wherein the aggregating conditions is heating or the addition of anaggregating excipient.
 54. The method of claim 52, wherein theaggregating excipient is aluminum hydroxide.
 55. A vaccine compositioncomprising an isolated alpha (2→8) or (2→9) oligosialic acid derivativehaving (i) a degree of polymerization of about 2-20, (ii) an IC50 ofless than about 0.1 μg/ml for inhibiting SEAM 2, SEAM 3 or DA2 antibodybinding to dodecylamine N-propionyl NmB polysialic acid or N-propionylNmB polysialic acid, and (iii) a non-reducing end de-N-acetyl residuethat is resistant to degradation by exoneuraminidase.
 56. The vaccinecomposition of claim 55, wherein said alpha (2→8) or (2→9) oligosialicacid derivative comprises one or more N-trichloroacetyl sialic acidresidues.
 57. The vaccine composition of claim 55, wherein said alpha(2→8) or (2→9) oligosialic acid derivative comprises one or moreN-propionyl sialic acid residues.
 58. The vaccine composition of claim55, wherein said alpha (2→8) or (2→9) oligosialic acid derivative has adegree of polymerization of about 2-10.
 59. The vaccine composition ofclaim 55, wherein said alpha (2→8) or (2→9) oligosialic acid derivativehas a degree of polymerization of about 2-6.
 60. The vaccine compositionof claim 55, wherein said alpha (2→8) or (2→9) oligosialic acidderivative is selected from the group consisting of dimer, trimer andtetramer.
 61. The vaccine composition of claim 55, wherein said alpha(2→8) or (2→9) oligosialic acid derivative comprises a conjugate. 62.The vaccine composition of claim 55, wherein said conjugate comprises asa first molecule said alpha (2→8) or (2→9) oligosialic acid derivativeconjugated to a second molecule comprising an immunomodulator.
 63. Thevaccine composition of claim 62, wherein said immunomodulator is a toxinor derivative thereof.
 64. The vaccine composition of claim 63, whereinsaid toxin or derivative thereof is tetanus toxoid.
 65. The vaccinecomposition of claim 55, wherein said oligosialic acid derivative saidis an alpha (2→8) oligosialic acid derivative selected from the groupconsisting of NPrSia-TT, OS-TT, and TcAc-TT.
 66. A vaccine compositioncomprising an isolated alpha (2→8) or (2→9) oligosialic acid derivativehaving (i) a degree of polymerization of about 2-20, (ii) a de-N-acetylsialic acid content of about 50% to 98%, and (iii) a non-reducing endde-N-acetyl residue that is resistant to degradation byexoneuraminidase.
 67. The vaccine composition of claim 66, wherein saidalpha (2→8) or (2→9) oligosialic acid derivative has a de-N-acetylsialic acid content of about 88% to 98%.
 68. The vaccine composition ofclaim 66, wherein said alpha (2→8) or (2→9) oligosialic acid derivativehas a degree of polymerization of about 2-10.
 69. The vaccinecomposition of claim 66, wherein said alpha (2→8) or (2→9) oligosialicacid derivative has a degree of polymerization of about 2-6.
 70. Thevaccine composition of claim 66, wherein said alpha (2→8) or (2→9)oligosialic acid derivative is selected from the group consisting ofdimer, trimer and tetramer.
 71. The vaccine composition of claim 66,wherein said alpha (2→8) or (2→9) oligosialic acid derivative vaccinecomprises a conjugate.
 72. The vaccine composition of claim 67, whereinsaid conjugate comprises as a first molecule said alpha (2→8) or (2→9)oligosialic acid derivative conjugated to a second molecule comprisingan immunomodulator.
 73. The vaccine composition of claim 72, whereinsaid immunomodulator is a toxin or derivative thereof.
 74. The vaccinecomposition of claim 73, wherein said toxin or derivative thereof istetanus toxoid.
 75. The vaccine composition of claim 74, wherein saidoligosialic acid derivative said is the alpha (2→8) oligosialic acidderivative DeNAc-TT.